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WO2018185735A1 - Combinaison antimicrobienne - Google Patents

Combinaison antimicrobienne Download PDF

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Publication number
WO2018185735A1
WO2018185735A1 PCT/IB2018/052437 IB2018052437W WO2018185735A1 WO 2018185735 A1 WO2018185735 A1 WO 2018185735A1 IB 2018052437 W IB2018052437 W IB 2018052437W WO 2018185735 A1 WO2018185735 A1 WO 2018185735A1
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WO
WIPO (PCT)
Prior art keywords
microbial
polynucleotide
seq
aptamer
combination
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PCT/IB2018/052437
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English (en)
Inventor
Darren John Day
Jennifer Pamela SOUNDY
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Victoria Link Limited
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Publication of WO2018185735A1 publication Critical patent/WO2018185735A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/38—Silver; Compounds thereof
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates generally to anti-microbial agents (AMA) having bacteriostatic and/or bactericidal properties, compositions and combinations comprising an AMA, and the use of such compositions, combinations and/or AMAs to inhibit the growth and/or proliferation of microorganisms, in the manufacture of medicaments for treating microbial infection, and/or for treating microbial infection.
  • AMA anti-microbial agents
  • anti-microbial resistance threatens the effective prevention and treatment of an ever- increasing range of infections caused by bacteria, parasites, viruses and fungi.
  • the WHO considers anti-microbial resistance to be an increasingly serious threat to global public health and calls on all government sectors and society in general to act, noting that anti-microbial resistance is present in all parts of the world, and that new resistance mechanisms emerge and spread globally. For example, there are high proportions of antibiotic resistance in bacteria that cause common infections (e.g.
  • MRSA methicillin-resistant Staphylococcus aureus
  • Pseudomonas aeruginosa is a clinically important, opportunistic, gram- negative bacterium that is responsible for a wide variety of severe hospital-acquired infections. It largely effects immune-compromised patients, particularly those with cystic fibrosis, as well as causing infections in burn wounds, and forming biofilms on implanted devices such as urinary catheters and heart stents.
  • P. aeruginosa is intrinsically resistant to many anti-microbial agents due to the low permeability of its outer membrane, and upregulation of multidrug efflux pump systems [1]. It can also acquire resistance mechanisms through horizontal gene transfer and chromosomal mutations.
  • Biofilms are a matrix of excreted exopolysaccharides, proteins and DNA that is formed over the colonised bacterial population which protects the bacteria inside from antibiotics [2]. These biofilm bacteria have adaptive changes in gene expression and shift to a metabolically less active state, making the infections harder to clear and are responsible for the recalcitrance of disease.
  • microorganims and/or in the the manufacture of medicaments for treating microbial infections and/or in methods of treating microbial infections, and/or to at least provide the public with a useful choice.
  • the invention in a first aspect, relates to an anti-microbial combination comprising a polynucleotide comprising a targeting component and an anti-microbial component, wherein the targeting component specifically binds a target molecule or cell, and wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent.
  • AMC an anti-microbial component comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or a polynucleotide that chelates at least one metal ion, or both, and ii) an anti-microbial agent.
  • the invention relates to a combination of formula II: AptPA-AMC wherein
  • AptPA is an aptamer or functional fragment thereof that specifically binds a Pseudomonas aeruginosa cell
  • AptPA is an aptamer or functional fragment thereof that specifically binds a Pseudomonas aeruginosa cell
  • the invention in another aspect relates to a method of making an anti-microbial combination comprising : a) synthesizing a polynucleotide comprising a targeting component comprising a polynucleotide that specifically binds to a target molecule or cell, and an anti- microbial component comprising a nucleic acid sequence that forms a metal nanocluster with at least three metal atoms, or is a nucleic acid sequence that chelates at least one metal ion, or both, and b) associating the polynucleotide synthesized in a) with at least one antimicrobial agent to make the anti-microbial combination.
  • the invention relates to an anti-microbial combination as described herein made by a method as described herein.
  • an anti-microbial component comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or a polynucleotide structure that chelates at least one metal ion, or both, and ii) an anti-microbial agent.
  • the invention in another aspect relates to a composition
  • a composition comprising an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, or a combination of formula II or Ila as described herein and a carrier, diluent or excipient.
  • the invention relates to the use of an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, a combination of formula II or Ila, or a composition as described herein, in the manufacture of a medicament for treating microbial infection.
  • a method of treating a microbial infection comprising administering an anti-microbial combination, an anti-microbial component, a combination of formula I, a combination of formula II or Ila, or a composition as described herein to a subject in need thereof.
  • the invention relates to an anti-microbial combination, an antimicrobial component, a combination of formula I, a combination of formula II or Ila, or a composition as described herein for use in treating, or when used to treat, microbial infection.
  • the invention relates to the use of an anti-microbial component as described herein to inhibit the growth and/or proliferation of at least one bacterial species, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion.
  • the invention relates to the use of an anti-microbial component as described herein in the manufacture of a medicament for treating microbial infection, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion.
  • the invention in another aspect relates to a method of inhibiting the growth and/or proliferation of at least one bacterial species comprising contacting the at least one bacterial species with an anti-microbial component as described herein, wherein the anti-microbial component comprises a polynucleotide in association with an antimicrobial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion.
  • the invention in another aspect relates to a method of treating a microbial infection comprising administering an anti-microbial component to a subject in need thereof, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion.
  • the invention in another aspect relates to an anti-microbial component for use in inhibiting the growth and/or proliferation of at least one bacterial species treating, or when used to treat, microbial infection, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion.
  • the invention relates to a polynucleotide selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43.
  • the invention relates to the use of an anti-microbial component to inhibit the growth and/or proliferation of at least one bacterial species
  • the anti-microbial component consists essentially of a polynucleotide structure in association with an anti-microbial agent, wherein the polynucleotide structure comprises an i-motif structure, wherein the anti-microbial agent is at least one metal ion, preferably at least one Ag + , wherein the imotif structure comprises a single nucleic acid strand that forms the i- motif structure or at least two, preferably four, nucleic acid strands that form the imotif structure, wherein the nucleic acid strand or strands that form the i-motif structure comprise a nucleic acid sequence comprising at least one, preferably at least two, preferably four poly C tracts (pCts), wherein the bacterial species is selected from the group consisting of Pseudomonas aeruginosa, Salmonella enterica serovar
  • FIG. 1 Tuneable fluorescence of DNA scaffolded silver nanoclusters.
  • the sequence of the DNA scaffold for AgNC synthesis plays a role in the emitted fluorescence of the clusters. Displayed in this figure is red, orange, yellow and green fluorescence (from left to right) of AgNCs.
  • FAM labelled aptamer left to right -10 nM, 50 nM, 100 nM, 250 nM
  • JN27 black, solid line
  • JN27.SH grey, solid line
  • aeruginosa PA692 aeruginosa PA692.
  • Cells were washed, re-suspended in buffer and analysed by flow cytometry to look for an increase in median fluorescence compared to bacteria with no aptamer (black dotted), and a difference between the two aptamers. Increased binding of the FAM labelled aptamers results in a right shift in the histograms.
  • Figure 4. Predicted aptamer secondary structures.
  • Figure 5. Specificity of aptamer binding to Pseudomonas aeruginosa species compared with other bacterial species. FAM-labelled aptamer (250 nM) was incubated with P. aeruginosa strains ( ⁇ 692 and PAOl) or other bacterial species (Salmonella enterica, Klebsiella pneumoniae, Enterobacter cloacae, Listeria innocua and
  • FIG 8. Thermal stability of JN21-NC fluorescence.
  • the emission spectrum of aptamer in MES buffer pH6.5 was recorded at 10°C and the relative fluorescence intensity set to 100%. The temperature was then raised in 5°C increments and the emission spectrum recorded and the relative intensity determined (grey bars). The experiment was repeated with fresh aptamer except after each temperature jump the sample was cooled to 10°C and the fluorescence intensity determined. The recovered fluorescence expressed as a percentage of the initial fluorescence is shown by the black bars.
  • Figure 9. CD spectra of JN 21 split and JN 17 3'.
  • FIG. 10 Fluorescence spectra of aptamer-AgNC after addition of Ag + .
  • the fluorescence emission (solid line) and excitation (dashed line) spectrum of JN 17 was recorded (black) prior to the addition of a 6-molar excess of silver nitrate (grey). Chelation of extra silver shifts the emission from the red into the green part of the visible spectrum.
  • FIG. 1 Antibacterial activity of Aptamer-AgNCs. Growth assays showing antibacterial activity of Aptamer-AgNCs. An exponentially growing culture of P.
  • aeruginosa PA692 was diluted such that 2xl0 5 cfu were present in 150 ⁇ _ of growth media, then aptamers were added to a final concentration of between 10 ⁇ and 78 nM as indicated in the legend. Growth was monitored by measuring the increase in OD 600 at 5 minute intervals for 20 hrs. Figure 12. Bacterial titre (cfu) as a percentage of starting inoculum after 20 hr exposure to 10 ⁇ aptamer-AgNC. Bacterial titre (cfu) as a percentage of starting inoculum after 20 hr exposure to 10 ⁇ aptamer-AgNC. P. aeruginosa was culture and treated with aptamer as described in Figure 11. After 20hrs growth, the number of viable cells was enumerated by plating onto agar and culturing overnight. The number of cfu present as a percentage of the initial inoculum size is presented.
  • FIG. 14 Bacterial titre (cfu) as a percentage of starting inoculum after 20 hr exposure to 2.5 ⁇ aptamer- Ag + - P. aeruginosa was cultured and treated with aptamer as described in Figure 13. After 20hrs growth, the number of viable cells was enumerated by plating onto agar and culturing overnight. The number of cfu present as a percentage of the initial inoculum size is presented.
  • FIG. 15 Bacterial growth on agar plates after exposure to aptamer-Ag + .
  • Bacteria were cultured as described in Figure 13 and were treated with 2.5 ⁇ aptamer-Ag + for 20 hrs. Undiluted samples (100 ⁇ ) were plated directly and grown overnight.
  • the JN27 5' Ag + and t21lpl7 3'Ag + treated cultures had less than 3 colonies present compared with the untreated control that was completely overgrown forming a bacterial lawn. Individual colonies can be seen on the JN 17 3' Ag + and JN21 split Ag + treated plates.
  • FIG. 16 Growth assays showing synergy between aptamer-AgNC and ciprofloxacin action.
  • P. aeruginosa was cultured and treated with aptamer as described in Figure 11 and treated with either no aptamer (control) or 1 ⁇ or 5 ⁇ aptamer-NC, in the presence or absence of 0.5 ⁇ g/ml ciprofloxacin. Growth was monitored by measuring the increase in OD 600 at 5 minute intervals for 20 hrs.
  • FIG. 17 The antibacterial activity of aptamer-Ag + against different strains of P. aeruginosa. Growth curves showing antibacterial activity of 2.5 ⁇ aptamer- Ag + against different strains of P. aeruginosa. Different laboratory strains and clinical isolates of P. aeruginosa were cultured as described in Figure 13 and were treated with 2.5 ⁇ of aptamer-Ag + . Untreated control (squares) and treatment with the equivalent amount of silver nitrate (2.5 ⁇ , triangles) are presented for comparison. Growth was monitored by measuring the increase in OD 600 at 5 minute intervals for 20 hrs.
  • Figure 18 Kaplan Meier survival analysis of Galleria mellonella larvae infected with P. aeruginosa then treated with aptamer-AgNC or aptamer- Ag + .
  • Galleria mellonella survival curves for larvae treated with aptamer-AgNC and aptamer- Ag + Larvae were infected with approximately 20 cfu of P. aeruginosa then treated with 10 ⁇ of the indicated concentration of aptamer-AgNC or aptamer- Ag + 30 min later. Survival was monitored at 1 hr intervals.
  • Figure 19 Propidium iodide (PI) uptake following treatment with aptamer- AgNC or aptamer-Ag + .
  • aeruginosa was treated with 5 ⁇ of aptamer-AgNC or aptamer-Ag + then stained with propidium iodide and analysed by flow cytometry to identify depolarized or dead cells. Depolarization of death causes an increasing right shift in the histogram.
  • Figure 20 Time course for killing of P. aeruginosa with aptamer-Ag + .
  • the table shows the number of cfu obtained after plating an inoculum containing 2xl0 5 cfu following treatment with 1 ⁇ JN21 split Ag + , after 1 and 3 hrs of treatment.
  • Binding of the seventh SELEX round was assessed relative to the starting library (light grey) to P. aeruginosa (A) and E. coli (B). The fluorescence of unstained cells is indicated in black.
  • the seventh SELEX round shows increased binding to P. aeruginosa (right shift in the histogram) relative to the starting library, but both the starting library and the seventh SELEX round bind equally poorly to E. coli.
  • FIG 22 Aptamer dissociation constants (Kd)
  • Figure 23 Specificity of aptamer binding to Pseudomonas aeruginosa species compared with other bacterial species.
  • FAM-labelled aptamer 250 nM was incubated with P. aeruginosa strains ( ⁇ 692 and PAOl) or other bacterial species (Salmonella enterica, Klebsiella pneumoniae, Enterobacter cloacae, Listeria innocua and Escherichia coli). Cells were washed, re-suspended in PBS and analysed by flow cytometry to measure the relative fluorescence intensity (arbitary fluorescence units, AFU) due to bound aptamer. All aptamer candidates preferentially bound the P.
  • FIG. 24 Antibacterial activity of aptamer chelated Ag + . LB was inoculated with 2xl0 5 CFU of P. aeruginosa strain PA692 and treated with aptamer-Ag + s from 10 ⁇ and 78 nM as indicated in the legend. Growth was monitored by measuring the increase in OD 600 at 5 minute intervals for 20 hrs.
  • LB was inoculated with P. aeruginosa strain PA692 and either untreated (black solid), or treated with 2.5 ⁇ of JN21-AgNC in which the AgNC forming sequence was either 5' (dark grey solid), 3' (light grey solid), 5' with T7 spacer (black dashed), 3' with T7 spacer (dark grey dashed), or split (light grey dashed).
  • FIG. 26 Antibacterial activity of aptamer-NCs.
  • LB was inoculated with 2xl0 5 CFU of P. aeruginosa strain PA692 and treated with aptamer-AgNCs from 10 ⁇ and 78 nM as indicated in the legend. Growth was monitored by measuring the increase in OD 600 at 5 minute intervals for 20 hrs.
  • FIG. 27 Circular dichroism spectra showing i-motif formation upon titration with Ag + . Spectra show the formation of a trough at about260 nm upon additon of excess silver ions as illustrated for the graph labelled Full iMotif. The indicated spectral changes are characteristic of i-motif formation.
  • FIG 28 Aptamers target delivery of AgNCs.
  • P. aeruginosa strain PA692 was treated with 2.5 ⁇ AgNC (NC, dark grey dashed) or 2.5 ⁇ aptamer-AgNC and growth compared to no treatment control (black solid).
  • JN27 5'-AgNC dark grey solid
  • JN21 split-AgNC light grey solid
  • St21Lpl7 split-AgNC black dashed.
  • the untargeted NC has some antimicrobial activity but conjugation to the aptamer sequences enhances the antibacterial activity resulting in a shift to the right in the graphs.
  • Figure 29 The untargeted NC has some antimicrobial activity but conjugation to the aptamer sequences enhances the antibacterial activity resulting in a shift to the right in the graphs.
  • the control plate (Ctrl) that was not treated with aptamer-NC is an overgrown lawn of bacteria.
  • individual colonies are visble with 5 ⁇ treatment and no colonies with 10 ⁇ treatement.
  • Fo JN21 AgNC the plate shows an overgrown lawn at 5 ⁇ treatment and individual colonies at 10 ⁇ treatment.
  • Figure 30 Agar plates showing remaining PA692 after 20 hours treatment with 0, 2.5, 5 or 10 ⁇ aptamer-Ag + .
  • the control plate (Ctrl) that was not treated with aptamer-AgiM is an overgrown lawn of bacteria.
  • JN27 AgiM, JN21 AgiM and St21Lpl7 AgiM a few colonies are visible at 2.5 ⁇ treatment and no colonies at 5 ⁇ or 10 ⁇ treatment.
  • Figure 31 Growth assays showing synergy between aptamer-AgNC and ciprofloxacin action.
  • P. aeruginosa was cultured and treated with either no aptamer (control), 1 ⁇ or 5 ⁇ aptamer-AgNC, in the presence or absence of 0.5 ⁇ g/ml ciprofloxacin. Growth was monitored by measuring the increase in OD 600 at 5 minute intervals for 20 hrs. Treatment with aptamer-AgNC and ciprofloxacin together inhibited growth to a greater extent than the sum of the individual treatments.
  • FIG 32 The antibacterial activity of aptamer-Ag + against different strains of P. aeruginosa. Different laboratory strains and clinical isolates of P. aeruginosa were cultured as described in Figure 17 and were treated with 2.5 ⁇ of aptamer-Ag + . Untreated control (black solid) and treatment with the equivalent amount of silver nitrate (2.5 ⁇ , light grey dashed) are presented for comparison. Growth was monitored by measuring the increase in OD 600 at 5 minute intervals for 20 hrs. All aptamer-Ag + conjugates prevented growth while the same concentration of silver nitrate slowed growth but was much less effective than the aptamer-Ag + .
  • FIG 33 Time-to-kill assay for aptamer-AgNCs.
  • P. aeruginosa strain PA692 was resuspended in MES buffer and treated with 5 ⁇ or 10 ⁇ aptamer-AgNC and the number of viable CFU remaining at each time point determined by agar plating. The aptamer-AgNC cause rapid killing of the bacteria.
  • Figure 34 Time-to-kill assay for aptamer-Ag + .
  • P. aeruginosa strain PA692 was resuspended in MES buffer and treated with 1 ⁇ or 2.5 ⁇ aptamer-Ag + and the number of viable CFU remaining at each time point determined by agar plating.
  • aptamer-Ag + cause rapid killing of the bacteria.
  • Figure 35 Comparison between dose dependent antimicrobial effects of aptamer-AgNC and aptamer-Ag + . The fold increase in lag phase compared to control growth was compared for each concentration of treatment with aptamer-AgNC or aptamer-Ag + , as determined by the growth assays in figure 24 and 26.
  • Figure 36 Aptamer-AgNCs depolarise P. aeruginosa cells after 10 min and 1 hr of treatment. P.
  • aeruginosa strain PA692 was treated with either, 1 ⁇ g/mL ciprofloxacin, 5 or 10 ⁇ aptamer-AgNC, or not at all (no treatment) for 10 min and 1 hr. Cells were then stained with 1.5 ⁇ g/mL propidium iodide (PI) and the fraction of high PI fluorescing (permeable cells) cells recorded. The positive control are cells killed with isopropanol.
  • PI propidium iodide
  • FIG. 37 Aptamer-Ag + s depolarise P. aeruginosa cells after 10 min and 1 hr of treatment.
  • P. aeruginosa strain PA692 was treated with no treatment, or a 2-fold dilution series of aptamer-Ag + from 5 ⁇ to 0.078 ⁇ , for 10 min or 1 hr then stained with propidium iodide (PI, 1.5 ⁇ g/mL) and the high PI fluorescing (permeable cells) population recorded.
  • PI propidium iodide
  • Figure 38 Kaplan Meier survival analysis of Galleria mellonella larvae infected with P. aeruginosa then treated with aptamer-AgNC or aptamer- Ag + .
  • Larvae were infected with approximately 10 CFU of P. aeruginosa PAOl .
  • Panel A+B show larvae infected with PAOl as above and then treated with 10 ⁇ of each aptamer-AgNC and the survival tracked for 24 hours as two independent experiments.
  • Figure 39 Dose response graphs of PAOl and isogenic MexB efflux pump mutant after treatment with ciprofloxacin or aptamer-AgNCs. Graphs show growth of the bacteria at each treatment concentration as a percentage of growth compared to no treatment control. Panel A shows the difference in IC50 between P. aeruginosa strain PAOl and MexB when treated with ciprofloxacin. Panels B-D show no significant difference in IC50 between PAOl and MexB treated with aptamer-AgNCs.
  • Figure 40 Dose response graphs of PAOl and isogenic MexB efflux pump mutant after treatment with ciprofloxacin or aptamer-Ag + s. Graphs show growth of the bacteria at each treatment concentration as a percentage of growth compared to no treatment control. Panel A shows the difference in IC50 between P. aeruginosa strain PAOl and MexB when treated with ciprofloxacin. Panels B-D show no significant difference in IC50 between PAOl and MexB treated with aptamer-Ag + s.
  • Figure 41 Changes to an immature biofilm after treatment with aptamer-Ag + or ciprofloxacin.
  • Biofilms were grown for 20 hours from PA692 and treated with 5 ⁇ aptamer-Ag + or ciprofloxacin once for 6 hours (panels A+B) or twice for 6 then 17 hours (panels C+D).
  • Panels A+C show the growth of planktonic cells shed from the biofilm during antimicrobial challenge
  • panels B+D show the reduction in biofilm biomass after treatments.
  • FIG. 42 Changes to a mature biofilm after treatment with aptamer-Ag + or ciprofloxacin. Biofilms were grown for 44 hours from P. aeruginosa strain PA692 and treated with 5 ⁇ aptamer-Ag + or ciprofloxacin once for 6 hours. Panel A shows planktonic growth of cells shed from the biofilm during treatment. Panel B shows the reduction in biofilm biomass after treatment.
  • FIG 43 Antibacterial activity of i-motif2 (SEQ ID NO:38) against various bacterial strains. Growth assays showing antibacterial activity of i-motif2. An exponentially growing culture of each bacterial strain was diluted such that 2xl0 5 cfu were present in 150 ⁇ of growth media then Ag + stabilised i-motif2 was prepared and added to a final concentration of between 2.5 ⁇ and 0.62 ⁇ as indicated in the legend. Growth was monitored by measuring the increase in OD 600 at 5 minute intervals for 17 hrs.
  • FIG 44 Antibacterial activity of a series of i-motif sequences against P. aeruginosa strain PA692. Growth assays showing antibacterial activity of various i- motif sequences. An exponentially growing culture of PA692 was diluted such that 2xl0 5 cfu were present in 150 ⁇ of growth media then Ag + stabilised I-motifs were prepared and added to a final concentration of between 5 ⁇ and 1.25 ⁇ as indicated in the legend . Growth was monitored by measuring the increase in OD 600 at 5 minute intervals for 17 hrs.
  • FIG. 45 Antibacterial activity of a series of i-motif sequences against P. aeruginosa strain PAOl. Growth assays showing antibacterial activity of various i- motif sequences. An exponentially growing culture of PA01 was diluted such that 2xl0 5 cfu were present in 150 ⁇ of growth media then Ag + stabilised I-motifs were prepared and added to a final concentration of between 5 ⁇ and 1.25 ⁇ as indicated in the legend . Growth was monitored by measuring the increase in OD 600 at 5 minute intervals for 17 hrs.
  • Figure 46 Circular dichroism spectra showing Ag + stabilised i-motif formation upon addition of Ag + of various predicted i-motif forming
  • Spectra show the formation of a trough at about 260 nm and a peak at 290 nm upon addition of excess silver ions.
  • the indicated bathochromic shift in spectral properties are characteristic of i-motif formation.
  • Figure 47 Antibacterial activity of a series of i-motif sequences against S. aureus. Growth assays showing antibacterial activity of various i-motif sequences. An exponentially growing culture of S. aureus was diluted such that 2xl0 5 cfu were present in 150 ⁇ of growth media then Ag + stabilised i-motifs were prepared and added to a final concentration of between 5 ⁇ and 1.25 ⁇ as indicated in the legend. Growth was monitored by measuring the increase in OD 600 at 5 minute intervals for 17 hrs.
  • FIG 48 Antibacterial activity of I-motif2 (SEQ ID NO: 38) against various bacterial strains and synergy with co-treatment with 2 mM EDTA. Growth assays showing antibacterial activity of i-motif2 and EDTA. Exponentially growing cultures of bacteria were diluted such that 2xl0 5 cfu were present in 150 ⁇ of growth media then Ag + stabilised i-motif2 was prepared and added to a final concentration of 1.25 ⁇ with or without 2 mM EDTA as indicated in the legend. Growth was monitored by measuring the increase in OD 600 at 5 minute intervals for 17 hrs.
  • Figure 49 Antibacterial activity of half i-motif 1 (SEQ ID NO: 39) against S.
  • aureus from 0.078 ⁇ to 1.25 ⁇ and synergy with co-treatment with a range of concentrations of EDTA.
  • Growth assays showing antibacterial activity of half i-motifl and EDTA. Exponentially growing cultures of S. aureus were diluted such that 2xl0 5 cfu were present in 150 ⁇ of growth media then Ag + stabilised half i-motifl was prepared and added to a final concentration range of between 1.25 ⁇ and 0.078 ⁇ , with 0.1, 0.15, 0.2, 0.5, or 0.75 mM EDTA as indicated in the legend. Growth was monitored by measuring the increase in OD 600 at 5 minute intervals for 17 hrs.
  • fragment as used herein is used interchangeably with the term “functional fragment” and means the same thing.
  • a functional fragment thereof when used in reference to a polynucleotide as described herein refers to that portion of the polynucleotide that is required for the activity of that polynucleotide, wherever that activity may be, and which could not reasonable be expected to have occurred by random chance.
  • a functional fragment thereof means that portion of the polynucleotide that is required for specific binding.
  • a targeting component as used herein with reference to a polynucleotide or functional fragment thereof means a polynucleotide or functional fragment thereof that specifically binds a target molecule or cell.
  • a targeting component of an anti-microbial combination as described herein comprises, consists essentially of, or consists of an aptamer or functional fragment thereof.
  • a high binding affinity means that on average, the binding affinity of an aptamer that binds to a given Gram negative bacterial species is at least 10 ⁇ , preferably at least 5 ⁇ , preferably at least 2 ⁇ , preferably at least 0.5 ⁇ .
  • a skilled person in the art using suitable conditions as described herein and known in the art can employ a "targeting component" as described herein to specifically bind to a target molecule or cell to distinguish that target molecule or cell from amongst a population of non-target molecules or cells.
  • the basis of this determination is the high binding affinity that a "targeting component", and particularly an aptamer or functional fragment thereof as described herein, has for a target molecule or cell as compared to a non-target molecule or cell.
  • aptamer means to a
  • polynucleotide or functional fragment thereof that binds, preferably that specifically binds, a target molecule or cell without the formation of canonical Watson-Crick complementary base pairs between the nucleotide residues in the aptamer and the residues of a target molecule to which the aptamer binds.
  • canonical Watson-Crick complementary base pairs between the nucleotide residues in the aptamer and the residues of a target molecule to which the aptamer binds.
  • internal Watson- Crick base pairing within the aptamer may be present, specific binding between an aptamer and a target molecule or cell is based on the three dimensional structure of the aptamer and target, and is selected for in the design and evolution of the aptamer.
  • aptamer or functional fragment of an aptamer as described herein means that the primary basis for the aptamer or functional fragment thereof binding to the target molecule or cell does not involve the formation of complementary nucleotide base pairs between the aptamer and the target molecule or cell.
  • nucleotide sequence of an aptamer may include base pairs that are not required for specific binding of the aptamer to a given target, and that smaller fragments of an aptamer, even fragments having below 50% sequence identity may still be capable of effectively binding to a target (Alsager, Omar A., et al. "Ultrasensitive Colorimetric Detection of 173-Estradiol : The Effect of Shortening DNA Aptamer Sequences.” Analytical chemistry 87.8 (2015) : 4201-4209).
  • an antimicrobial component or "a polynucleotide” that is “in association” or “associated with” an anti-microbial agent or with reference to “associating the" "anti-microbial component” or “polynucleotide” with an “anti-microbial agent” means that the antimicrobial agent is connected to the "anti-microbial component” or “polynucleotide” by at least one chemical bond.
  • At least one chemical bond means at least one covalent, coordination, or ionic bond.
  • That associates, under the appropriate conditions and similar
  • anti-microbial agent and “additional anti-microbial agent” as used herein refer to any agent that has microbiocidal and/or microbiostatic activity against a microorganism or microscopic lifecycle stage of an organism.
  • an "antimicrobial agent” and/or an “additional anti-microbial agent” have microbiocidal, preferably bactericidal activity.
  • anti-microbial agents that have activity against viruses, bacteria, protists and fungi.
  • Preferred anti-microbial agents used in the context of the present invention are anti-bacterial agents having bactericidal and/or bacteriostatic properties.
  • An anti-microbial agent can be an organic or inorganic.
  • inhibiting the growth and/or proliferation refers to a measureable reduction in the number of bacteria present, and/or in the duration of the bacterial presence or infection.
  • inhibiting the growth and/or proliferation is determined by comparative assay of the optical density at 600 nm over time, of a bacterial control culture vs. a bacterial culture treated with an antibacterial combination or composition as described herein.
  • conjugate and "conjugated” as used herein mean bound by at least one covalent bond.
  • a conjuggate will comprise at least two components joined together by at least one covalent bond.
  • complex and “complexed” as used herein means bound by at least one non-covalent chemical bond that may be a coordination bond or an ionic bond.
  • a “complex” will comprise at least two components joined together by at least one coordination bond or by at least one ionic bond or that are physically associated by other molecular interactions, such as but not limited to pi pi stacking.
  • polynucleotide scaffold refers to a polynucleotide sequence that links a targeting component as described herein to an antimicrobial agent as described herein.
  • a polynucleotide scaffold may be designed to minimize steric conflicts between the targeting component, or at least a portion of a targeting component, and an anti-microbial agent.
  • the polynucleotide scaffold also functions to stabilize nanociusters and /or retain bound metal ions.
  • the polynucleotide may enhance the specificity of the targeting component.
  • the polynucleotide scaffold interacts and/or associates with the targeting component and contributes to the binding of a target cell or molecule by the targeting component.
  • a polynucleotide scaffold may form secondary structures either alone or in combination with other nucleic acid residues in an anti-microbial combination as described herein.
  • the nucleic acid residues in the polynucleotide scaffold may interact with the nucleic acid residues in a targeting component.
  • the polynucleotide scaffold forms an i-motif, or at least a portion of an i-motif.
  • i-motif refers to a polynucleotide sequence that is cytosine rich whose core is arranged as two parallel duplexes that are inter-chelated in an anti- parallel manner.
  • An "i-motif” can be formed by one strand, or two strands or four strands spatially arranged to form the "i-motif" core.
  • the “i-motif” structure may be stabilised by acidic pH or bound metal ions such as silver or copper.
  • Polynucleotides adopting the "i-motif" structure can be identified on the basis of their spectroscopic properties, in particular their circular dichroism spectra, enhance fluorescence emission upon binding the dye neutral red, and UV difference spectra following thermal denaturation or metal ion binding.
  • the spectral properties of i-motif forming polynucleotides are well reported in the literature [23], [24], [25].
  • i-motif is used interchangeably herein with the term “i-motif structure” and means the same thing.
  • the terms “imotif”, “i-motif”, “i-motif structure” and grammatical variations thereof mean a polynucleotide or polynucleotides that is/ar silver stabilised to form an i-motif (also termed a “silver stabilized i-motif” herein) in which the polnucleotide(s) exist in free solution at neutral pH in a configuration that is not a fully folded i-motif.
  • Ag + alters the conformation of the polynucleotide(s) stabilizing the
  • the polynucleotide(s) to form a silver stabilised n i-motif.
  • the i-motif is formed from a single polynucleotide (i.e., a single nucleic acid strand) the addition of Ag + bends the polynucleotide into the fully folded i-motif.
  • silver nanocluster as used herein means a collection of 3 to 50 silver atoms (Ag) that is formed by the reduction of silver ions and stabilized by interaction with a polynucleotide scaffold.
  • the polynucleotide scaffold is a DNA scaffold.
  • silver ion as used herein means Ag + .
  • silica metal as used herein means Ag.
  • aptamer-Ag + as used herein means aptamers that form i-motifs that remain stably associated with Ag + .
  • statically significant refers to the likelihood that a result or relationship is caused by something other than random chance.
  • a result may be found to be statistically significant using statistical hypothesis testing as known and used in the art.
  • Statistical hypothesis testing provides a "P-value" as known in the art, which represents the probability that the measured result is due to random chance alone. It is believed to be generally accepted in the art that levels of significance of 5% (0.05) (e.g., the observed result occurs by chance less than 5% of the time) or lower are considered to be strongly statistically significant, and P values between 0.2 and 0.05 are considered to be weakly significant.
  • the terms “treat”, “treating” and “treatment” refer to therapeutic measures which reduce, alleviate, ameliorate, manage, prevent, restrain, stop or reverse microbial infection including the symptoms associated with or related to microbial infection.
  • the subject may show observable or measurable (statistically significant) decrease in one or more of the symptoms associated with or related microbial infection as known to those skilled in the art, as indicating improvement.
  • an "effective amount” as used herein means an amount effective to protect against, delay, reduce, stabilise, improve or treat microbial infection as known in the art, and/or as described herein.
  • an "effective amount" of a targeting component, an anti-microbial component and/or an anti-microbial combination as described is an amount that is sufficient to achieve at least a lessening of the symptoms associated with a microbial infection that is being or is to be treated or that is sufficient to achieve a reduction in microbial growth, or that is sufficient to increase in microbial susceptibility to other therapeutic agents or natural immune clearance.
  • an effective amount is an amount sufficient to achieve a statistically different result as compared to an untreated control.
  • a “formulation agent” as used herein refers to any compound or material that facilitates or optimizes the production, handling, storage, transport, application and/or persistence of a targeting component, an anti-microbial component, an anti-microbial combination, and or a composition of, or for use in the invention, but not limited thereto.
  • Subject as used herein is an animal, preferably a vertebrate animal or an
  • the vertebrate animal is a mammal.
  • the mammal includes human and non-human mammals including but not limited to cats, dogs, horses, cows, sheep, deer, mice, rats, primates (including gorillas, rhesus monkeys and chimpanzees), pigs, possums and other domestic farm or zoo animals, but not limited thereto.
  • the mammal is human.
  • the term "about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, "about 100" means from 90 to 110 and "about six" means from 5.4 to 6.6.
  • the present invention relates generally to the inventors unexpected identification of anti-microbial combinations that comprise at least one polynucleotide component in association with at least one anti-microbial agent.
  • the inventors have identified that a polynucleotide that specifically binds a molecule or cell can be combined with a polynucleotide that associates with an anti-microbial agent into an anti-microbial combination to deliver the anti-microbial agent directly to the target molecule or cell.
  • a polynucleotide that specifically binds a molecule or cell is termed a "targeting component" as described herein.
  • a polynucleotide that associates with an anti-microbial agent is termed an "anti-microbial component" as described herein.
  • an anti-microbial combination comprises a targeting component that is an aptamer or functional fragment thereof that specifically binds Pseudomonas aeruginosa and an anti-microbial component comprising a nucleic acid sequence encoding a polynucleotide scaffold that associates, under the appropriate conditions, with sufficient silver ions to form a DNA scaffolded silver nanocluster upon reduction
  • a targeting component that is an aptamer or functional fragment thereof that specifically binds Pseudomonas aeruginosa
  • an anti-microbial component comprising a nucleic acid sequence encoding a polynucleotide scaffold that associates, under the appropriate conditions, with sufficient silver ions to form a DNA scaffolded silver nanocluster upon reduction
  • the silver in the nanocluster in the anti-microbial combination is
  • an anti-microbial combination comprises a targeting component that is an aptamer or functional fragment thereof that specifically binds Pseudomonas aeruginosa and an anti-microbial component that is a polynucleotide scaffold that forms, or that forms part of, an i-motif where the i-motif associates, under the appropriate conditions, with at least one silver (Ag + ) or copper (Cu + , Cu ++ ) ion.
  • a targeting component as described herein particularly an aptamer or functional fragment thereof that specifically binds to a pathogenic bacterial cell, can potentiate the activity and/or efficacy of other anti-microbial agents.
  • the inventors have identified that the anti-microbial components, anti-microbial combinations and compositions described herein have unexpectedly high efficacy for killing bacteria, particularly Pseudomonas spp. bacteria.
  • a targeting component comprising a polynucleotide that specifically targets a molecule or cell
  • an anti-microbial component comprising o a nucleic acid sequence encoding a polynucleotide scaffold that
  • the inventors further believe that they are the first to identify that polynucleotide that forms, or that forms part of an i-motif, when complexed with at least one silver or copper ion, is effective therapeutically when used alone as an anti-microbial, particularly an anti-bacterial agent.
  • an antimicrobial component that is an i-motif-Ag + combination (also termed an "imotif alone") herein can be used to inhibit the growth and/or proliferation of
  • microorganisms particularly bacteria
  • therapeutically to treat microbial, particularly bacterial infection are disclosed. Additionally, the inventors disclose herein how combining such an anti-microbial component with a targeting component as described herein provides an effective anti-microbial combination.
  • DNA aptamers which bind with a high affinity and specificity to live biofilm derived P. aeruginosa cells. These aptamers or a functional fragment thereof may be used in or as a targeting
  • anti-microbial components as described herein to deliver anti-microbial components as described herein to the site of microbial, and particularly of Pseudomonas infection.
  • the anti-microbial components themselves, as described herein may be used alone in therapeutic applications that do not require specific targeting, for example, topical applications, but not limited thereto.
  • the targeting component of an anti-microbial combination as described herein may be an aptamer or a functional fragment thereof as described herein that specifically binds target bacteria, particularly Pseudomonas spp. bacteria, and more particularly Pseudomonas aeruginosa.
  • Aptamers show potential as new therapeutics against pathogenic bacteria. They have been shown to have bacteriostatic effects against Salmonella [3], and inhibitory properties against Mycobacterium [4]. Aptamers are short, single stranded DNA or RNA oligonucleotides that can be selected to a wide variety of microbial targets including viruses [5] and bacteria [6, 7]. Aptamers are made by a Systematic
  • aptamers are relatively non-immunogenic and non-toxic due to their small size, and can be rapidly chemically synthesised and modified, making them an interesting candidate for new drug development. Aptamers form stable secondary structures which allows them to bind to their target with high affinity and specificity, and potentially cause a therapeutic effect.
  • the aptamers described herein are made by chemical synthesis.
  • aptamers as described herein may be made by other means as known in the art, for example, by enzymatic synthesis in vitro or in vivo expression in a permissive cell, but not limited thereto. What is important is that based on the disclosure herein by the inventors of the primary nucleotide sequences of the aptamers as described herein, a skilled worker can make any of the aptamers as described herein as known in the art, including any aptamer or a functional fragment thereof having one or more modifications to that aptamer as described herein as would be appropriate in the context of the invention and/or for use in the present invention.
  • aptabiotics comprise bacterial species-specific polynucleotide aptamers or a functional fragment thereof, that specifically bind to bacteria, particularly Gram-negative bacteria, and that are complexed with antimicrobial components as described herein.
  • the anti-microbial components comprise polynucleotide scaffolds comprising silver nanoclusters (AgNC) .
  • the anti-microbial components comprise polynucleotide scaffolds that form, or that form part of, an i-motif.
  • an aptabiotic specifically binds live whole bacterial cells.
  • the live whole bacterial cells are Pseudomonas spp. cells, preferably P. aeruginosa cells.
  • aptabiotics can be used to deliver AgNCs and i-motif/ Ag + complexes to specifically targeted living bacteria to render such otherwise multi-drug resistant bacteria, susceptible to antibiotic action.
  • Aptabiotics, targeting components and/or anti-microbial components as described herein may have both intrinsic anti-microbial activity and synergistic activity with existing
  • fluoroquinolone antibiotics that are used clinically to treat pathogenic bacterial infections, particularly Pseudomonas spp. infections, particularly P. aeruginosa infections [10].
  • pathogenic bacterial infections particularly Pseudomonas spp. infections, particularly P. aeruginosa infections [10].
  • the therapeutic, anti-microbial use of aptabiotics, targeting components and/or antimicrobial components provides a generic approach for targeted anti-microbial therapy applicable to many pathogenic microorganisms, particularly bacteria.
  • the aptabiotics disclosed herein have synergistic activity generally with existing antibiotics that are used clinically, particularly with
  • Pseudomonas spp. infections particularly P. aeruginosa infections.
  • the anti-microbial activity of silver ions and silver nanoparticles is well reported [11- 13].
  • Silver ions are also able to synergistically increase the efficacy of many antibiotics [14], however, the medicinal use of silver has been limited as it has not been possible to restrict its toxicity to just bacterial cells at the infection site.
  • Aptabiotics are a novel therapeutic that specifically deliver a toxic dose of silver to target cell.
  • aptabiotics augment the activity of existing antibiotics, while the circumventing resistance mediated by multi-drug resistance efflux pumps, as neither the silver metal nor silver ions are extruded by theses pumps.
  • AgNCs are small clusters of silver atoms stabilised by polynucleotides, with a well-defined, small (3-50) number of silver atoms, that exhibit bright fluorescence. Disclosed herein are AgNCs (Fig. 1) with tuneable fluorescence properties that depend on the sequence of the associated polynucleotide scaffolding.
  • I-motif DNA structures have been used for various purposes.
  • an anti-microbial combination comprising an aptamer or functional fragment thereof as described herein can specifically bind a target molecule or cell, particularly a live target bacterial cell (including Pseudomonas aeruginosa) and deliver silver nanoclusters or silver ions that disrupt membrane integrity and cellular activities.
  • the anti-microbial combination described herein also inhibits growth of bacteria in liquid culture, and biofilm formation, and synergizes with antibiotics, particularly floroquinone antibiotics such as Ciprofloxacin, to enhance antibiotic efficacy.
  • antibiotics particularly floroquinone antibiotics such as Ciprofloxacin
  • silver nanoparticles colloidal silver
  • silver nano-coatings are used in medical implants, to the best of the inventor's knowledge, neither the targeted delivery of silver metal (Ag) in the form of nanoclusters or nanoparticles, nor the targeted delivery of silver ions (Ag + ) complexed to i-motif polynucleotide scaffolds, to bacteria, using aptamers has been previously reported.
  • the aptabiotics described herein are novel anti-microbial agents when used directly to inhibit the growth of a targeted microbial pathogen, (for example, P. aeruginosa but not limited thereto). Additionally and importantly, the aptabiotics as described herein may restore sensitivity to antibiotics of a targeted pathogen where that pathogen was previously resistant to those antibiotics) (for example, where the targeted pathogen is P.
  • aeruginosa but not limited thereto.
  • the invention relates to an anti-microbial combination comprising a polynucleotide comprising a targeting component and an anti-microbial component, wherein the targeting component specifically binds a target molecule or cell, and wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent.
  • Targeting component specifically binds a target molecule or cell, and wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent.
  • the targeting component comprises a polynucleotide or functional fragment thereof that specifically binds the target molecule or cell.
  • the targeting component consists essentially of a polynucleotide or functional fragment thereof that specifically binds the target molecule or cell. In one embodiment the targeting component consists of a polynucleotide or functional fragment thereof that specifically binds the target molecule or cell.
  • the targeting component comprises a polynucleotide or functional fragment thereof that is a single stranded nucleic acid and may be either single stranded RNA or DNA.
  • the polynucleotide or functional fragment thereof is DNA.
  • the targeting component comprises a polynucleotide or functional fragment thereof that comprises a ribonucleotide, deoxyribonucleotide, or other type of nucleic acid, or two or more different types of nucleic acids.
  • the targeting component comprises a polynucleotide or functional fragment thereof that comprises one or more modified bases, sugars, polyethylene glycol spacers or backbone modifications.
  • the targeting component comprises a polynucleotide or functional fragment thereof that comprises one or more 2' sugar modifications, such as a 2'-0- alkyl (e.g., 2'-0-methyl or 2'-0- methoxyethyl) or a 2'-fluoro modification, but not limited thereto.
  • the targeting component comprises a polynucleotide or functional fragment thereof that comprises a nucleic acid sequence that specifically binds the target molecule or cell.
  • the nucleic acid sequence is an
  • oligonucleotide In one embodiment the oligonucleotide is selected from the group consisting of an antisense oligonucleotides, shRNAs, siRNAs and aptamers.
  • the targeting component comprises an aptamer or a functional fragment thereof. In one embodiment the targeting component consists essentially of an aptamer or a functional fragment thereof. In one embodiment the targeting component consists of an aptamer or a functional fragment thereof.
  • the polynucleotide, aptamer or functional fragment thereof is about 120, about 115, about 110, about 105, about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, or about 10 nucleotides in length.
  • the polynucleotide, aptamer or functional fragment thereof is 120, 110, 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, or 10 nucleotides in length.
  • the polynucleotide, aptamer or functional fragment thereof is less than 120, less than 115, less than 110, less than 105, less than 100, less than 95, less than 90, less than 85, less than 80, less than 75, less than 70, less than 65, less than 60, less than 55, less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, or less than 10 nucleotides in length
  • the polynucleotide, aptamer or functional fragment thereof is about 110 to about 120, about 100 to about 110, about 90 to about 100, about 80 to about 90, about 70 to 80, about 60 to 70, about 50 to 60, about 40 to 50, about 30 to 40, about 20 to 30, or about 10 to 20 nucleotides in length.
  • the polynucleotide, aptamer or functional fragment thereof is 110 to 120, 100 to 110, 90 to 100, 80 to 90, 70 to 80, 60 to 70, 50 to 60, 40 to 50, 30 to 40, 20 to 30, or 10 to 20 nucleotides in length.
  • the polynucleotide, aptamer or functional fragment thereof is about 105 to about 115, about 95 to about 105, about 85 to about 95, about 75 to 85, about 65 to 75, about 55 to 65, about 45 to 55, about 35 to 45, about 25 to 35, or about 15 to 25 nucleotides in length.
  • the polynucleotide, aptamer or functional fragment thereof is 105 to 115, 95 to 105, 85 to 95, 75 to 85, 65 to 75, 55 to 65, 45 to 55, 35 to 45, 25 to 35, or 15 to 25 nucleotides in length. In some embodiments the polynucleotide, aptamer or functional fragment thereof is about 90 to about 80 or about 88 to about 82 or about 86 to about 84, or about 86, or about 85 or about 84 nucleotides in length, preferably 90 to 80 or 88 to 82 or 86 to 84, or 86, or 85 or 84 nucleotides in length.
  • the polynucleotide, aptamer or functional fragment thereof is about 40 to about 50 or about 42 to about 48 or about 44 to about 46, or about 43 or about 45 nucleotides in length, preferably 40 to 50 or 42 to 48 or 44 to 46 or 43 or 45 nucleotides in length.
  • the polynucleotide, aptamer or functional fragment thereof is about 30 to about 40 or about 32 to about 38 or about 34 to about 36, or about 32 or about 35 or about 36 or about 38 nucleotides in length, preferably 30 to 40 or 32 to 38 or 34 to 36, or 32 or 35 or 36 or 38 nucleotides in length.
  • the polynucleotide, aptamer or functional fragment thereof is about 20 to about 30, or about 22 to about 28 or about 24 to about 26 or about 24 or about 25 or about 26 nucleotides in length, preferably 20 to 30, or 22 to 28 or 24 to 26 or 24 or 25 or 26 nucleotides in length.
  • the polynucleotide, aptamer or functional fragment thereof may be any length polynucleotide, aptamer or functional fragment thereof that falls within the size parameters set out herein.
  • a polynucleotide, aptamer or functional fragment thereof may be about 86, about 85, about 76, about 61, about 54, about 43, about 38, about 32, or about 27 nucleotides in length or may be 84, 78, 70, 58, 43, 35, or 26 nucleotides in length. What is important is that the polynucleotide, aptamer or functional fragment thereof specifically binds the target molecule or cell.
  • the aptamer or functional fragment thereof comprises a polynucleotide having a least 70% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO: 1 to 30.
  • the aptamer or a functional fragment thereof comprises a polynucleotide having at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 1 to 30.
  • the aptamer or functional fragment thereof comprises a polynucleotide selected from the group consisting of SEQ ID NO: 1 to 30.
  • the aptamer or functional fragment thereof consists essentially of a polynucleotide having a least 70% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO: 1 to 30.
  • the aptamer or a functional fragment thereof consists essentially of a polynucleotide having at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 1 to 30.
  • the aptamer or functional fragment thereof consists essentially of a polynucleotide selected from the group consisting of SEQ ID NO: 1 to 30.
  • the aptamer or functional fragment thereof consists of a polynucleotide having a least 70% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO: 1 to 30.
  • the aptamer or a functional fragment thereof consists of a polynucleotide having at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 1 to 30.
  • the aptamer or functional fragment thereof consists of a polynucleotide selected from the group consisting of SEQ ID NO: 1 to 30.
  • the aptamer is selected from the group consisting of JN27 (SEQ ID NO: 1); JN08 (SEQ ID NO: 2); JN27.SH (SEQ ID NO: 3); JN27 5' NC (SEQ ID NO: 4); JN27 3' NC (SEQ ID NO: 5);JN27 split NC (SEQ ID NO: 6); JN08.SH (SEQ ID NO: 7); JN08 5' NC (SEQ ID NO: 8); JN08 3' NC (SEQ ID NO: 9); JN08 split NC (SEQ ID NO: 10); JN21.SH (SEQ ID NO: 11); JN21 5' NC (SEQ ID NO: 12); JN21 3' NC (SEQ ID NO: 13); JN21 split NC (SEQ ID NO: 14); JN 17.SH (SEQ ID NO: 15); JN 17 5' SH (SEQ ID NO: 16); JN 17 3' SH (SEQ ID NO: 17); JN 17 split NC (SEQ ID NO: 18); St21L
  • St21Lpl7 split NC SEQ ID NO: 22
  • Stl7Lp21 SEQ ID NO: 23
  • Stl7Lp21 5' NC SEQ ID NO: 24
  • Stl7Lp21 3' NC SEQ ID NO: 25
  • Stl7Lp21 split NC SEQ ID NO: 26
  • St08Lpl7 SEQ ID NO: 27
  • St08Lpl7 5' NC
  • St08Lpl7 3' NC
  • St08Lpl7 split NC SEQ ID NO: 30
  • the aptamer is a functional fragment of any one of JN27 (SEQ ID NO: 1); JN08 (SEQ ID NO: 2); JN27.SH (SEQ ID NO: 3); JN27 5' NC (SEQ ID NO: 4); JN27 3' NC (SEQ ID NO: 5);JN27 split NC (SEQ ID NO: 6); JN08.SH (SEQ ID NO: 7); JN08 5' NC (SEQ ID NO: 8); JN08 3' NC (SEQ ID NO: 9); JN08 split NC (SEQ ID NO: 10); JN21.SH (SEQ ID NO: 11); JN21 5' NC (SEQ ID NO: 12); JN21 3' NC (SEQ ID NO: 13); JN21 split NC (SEQ ID NO: 14); JN 17.SH (SEQ ID NO: 15); JN 17 5' SH (SEQ ID NO: 16); JN 17 3' SH (SEQ ID NO: 17); JN 17 split NC (SEQ ID NO: 18);
  • St21Lpl7 (SEQ ID NO: 19); St21Lpl7 5' NC (SEQ ID NO: 20); St21Lpl7 3' NC (SEQ ID NO: 21); St21Lpl7 split NC (SEQ ID NO: 22); Stl7Lp21 (SEQ ID NO: 23);
  • Stl7Lp21 5' NC SEQ ID NO: 24
  • Stl7Lp21 3' NC SEQ ID NO: 25
  • Stl7Lp21 split NC SEQ ID NO: 26
  • St08Lpl7 SEQ ID NO: 27
  • St08Lpl7 5' NC SEQ ID NO: 28
  • St08Lpl7 3' NC SEQ ID NO: 29
  • St08Lpl7 split NC SEQ ID NO: 30.
  • JN 17 (SEQ ID NO: 1); JN 17-SH (SEQ ID NO: 2) ; JN21 (SEQ ID NO: 3); JN21-SH (SEQ ID NO: 4); JN27 (SEQ ID NO: 5) and JN27-SH (SEQ ID NO: 6).
  • the aptamer is about 86 nucleotides in length and comprises SEQ ID NO: 1 or a functional fragment thereof. In one embodiment, the aptamer is about 86 nucleotides in length and consists essentially of SEQ ID NO: 1 or a functional fragment thereof. In one embodiment, the aptamer is about 86 nucleotides in length and consists of SEQ ID NO: 1 or a functional fragment thereof. In one embodiment, the aptamer is 86 nucleotides in length and comprises SEQ ID NO: 1 or a functional fragment thereof. In one embodiment, the aptamer is 86 nucleotides in length and consists essentially of SEQ ID NO: 1 or a functional fragment thereof. In one embodiment, the aptamer is 86 nucleotides in length and consists of SEQ ID NO: 1 or a functional fragment thereof.
  • the aptamer is about 85 nucleotides in length and comprises SEQ ID NO: 2 or a functional fragment thereof. In one embodiment, the aptamer is about 85 nucleotides in length and consists essentially of SEQ ID NO: 2 or a functional fragment thereof. In one embodiment, the aptamer is about 85 nucleotides in length and consists of SEQ ID NO: 2 or a functional fragment thereof.
  • the aptamer is 85 nucleotides in length and comprises SEQ ID NO: 2 or a functional fragment thereof. In one embodiment, the aptamer is 85 nucleotides in length and consists essentially of SEQ ID NO: 2 or a functional fragment thereof. In one embodiment, the aptamer is 85 nucleotides in length and consists of SEQ ID NO: 2 or a functional fragment thereof.
  • the aptamer is about 43 nucleotides in length and comprises SEQ ID NO: 3 or a functional fragment thereof. In one embodiment, the aptamer is about 43 nucleotides in length and consists essentially of SEQ ID NO: 3 or a functional fragment thereof. In one embodiment, the aptamer is about 43 nucleotides in length and consists of SEQ ID NO: 3 or a functional fragment thereof.
  • the aptamer is 43 nucleotides in length and comprises SEQ ID NO: 3 or a functional fragment thereof. In one embodiment, the aptamer is 43 nucleotides in length and consists essentially of SEQ ID NO: 3 or a functional fragment thereof. In one embodiment, the aptamer is 43 nucleotides in length and consists of SEQ ID NO: 3 or a functional fragment thereof.
  • the aptamer is about 26 nucleotides in length and comprises SEQ ID NO: 7 or a functional fragment thereof. In one embodiment, the aptamer is about 26 nucleotides in length and consists essentially of SEQ ID NO: 7 or a functional fragment thereof. In one embodiment, the aptamer is about 26 nucleotides in length and consists of SEQ ID NO: 7 or a functional fragment thereof.
  • the aptamer is 26 nucleotides in length and comprises SEQ ID NO: 7 or a functional fragment thereof. In one embodiment, the aptamer is 26 nucleotides in length and consists essentially of SEQ ID NO: 7 or a functional fragment thereof. In one embodiment, the aptamer is 26 nucleotides in length and consists of SEQ ID NO: 7 or a functional fragment thereof.
  • the aptamer is about 45 nucleotides in length and comprises SEQ ID NO: 11 or a functional fragment thereof. In one embodiment, the aptamer is about 45 nucleotides in length and consists essentially of SEQ ID NO: 11 or a functional fragment thereof. In one embodiment, the aptamer is about 45 nucleotides in length and consists of SEQ ID NO: 11 or a functional fragment thereof.
  • the aptamer is 45 nucleotides in length and comprises SEQ ID NO: 11 or a functional fragment thereof. In one embodiment, the aptamer is 45 nucleotides in length and consists essentially of SEQ ID NO: 11 or a functional fragment thereof. In one embodiment, the aptamer is 45 nucleotides in length and consists of SEQ ID NO: 11 or a functional fragment thereof.
  • the aptamer is about 36 nucleotides in length and comprises SEQ ID NO: 15 or a functional fragment thereof. In one embodiment, the aptamer is about 36 nucleotides in length and consists essentially of SEQ ID NO: 15 or a functional fragment thereof. In one embodiment, the aptamer is about 36 nucleotides in length and consists of SEQ ID NO: 15 or a functional fragment thereof.
  • the aptamer is 36 nucleotides in length and comprises SEQ ID NO: 15 or a functional fragment thereof. In one embodiment, the aptamer is 36 nucleotides in length and consists essentially of SEQ ID NO: 15 or a functional fragment thereof. In one embodiment, the aptamer is 36 nucleotides in length and consists of SEQ ID NO: 15 or a functional fragment thereof.
  • the aptamer is about 38 nucleotides in length and comprises SEQ ID NO: 19 or a functional fragment thereof. In one embodiment, the aptamer is about 38 nucleotides in length and consists essentially of SEQ ID NO: 19 or a functional fragment thereof. In one embodiment, the aptamer is about 38 nucleotides in length and consists of SEQ ID NO: 19 or a functional fragment thereof.
  • the aptamer is 38 nucleotides in length and comprises SEQ ID NO: 19 or a functional fragment thereof. In one embodiment, the aptamer is 38 nucleotides in length and consists essentially of SEQ ID NO: 19 or a functional fragment thereof. In one embodiment, the aptamer is 38 nucleotides in length and consists of SEQ ID NO: 19 or a functional fragment thereof.
  • the aptamer is about 35 nucleotides in length and comprises SEQ ID NO: 23 or a functional fragment thereof. In one embodiment, the aptamer is about 35 nucleotides in length and consists essentially of SEQ ID NO: 23 or a functional fragment thereof. In one embodiment, the aptamer is about 35 nucleotides in length and consists of SEQ ID NO: 23 or a functional fragment thereof.
  • the aptamer is 35 nucleotides in length and comprises SEQ ID NO: 23 or a functional fragment thereof. In one embodiment, the aptamer is 35 nucleotides in length and consists essentially of SEQ ID NO: 23 or a functional fragment thereof. In one embodiment, the aptamer is 35 nucleotides in length and consists of SEQ ID NO: 23 or a functional fragment thereof.
  • the aptamer is about 32 nucleotides in length and comprises SEQ ID NO: 27 or a functional fragment thereof. In one embodiment, the aptamer is about 32 nucleotides in length and consists essentially of SEQ ID NO: 27 or a functional fragment thereof. In one embodiment, the aptamer is about 32 nucleotides in length and consists of SEQ ID NO: 27 or a functional fragment thereof.
  • the aptamer is 32 nucleotides in length and comprises SEQ ID NO: 27 or a functional fragment thereof. In one embodiment, the aptamer is 32 nucleotides in length and consists essentially of SEQ ID NO: 27 or a functional fragment thereof. In one embodiment, the aptamer is 32 nucleotides in length and consists of SEQ ID NO: 27 or a functional fragment thereof.
  • a polynucleotide, aptamer or functional fragment thereof of the invention may have intrinsic anti-microbial activity, particularly bacteriostatic or bactericidal activity.
  • intrinsic anti-microbial activity particularly bacteriostatic or bactericidal activity.
  • the inventors have also found that the intrinsic activity of the
  • polynucleotide, aptamer or functional fragment thereof may be potentiated by combining the polynucleotide, aptamer or functional fragment thereof with an antimicrobial component as described herein to form an anti-microbial combination of the invention.
  • the target molecule is a molecule on a cell.
  • the cell is a microorganism.
  • the microorganism is selected from the group consisting of fungi, viruses, bacteria, and protists.
  • the target microorganism is a living microorganism.
  • the living microorganism is comprised in a biofilm. In one embodiment the living
  • microorganism is a bacteria, preferably a Gram negative bacteria, preferably
  • Pseudomonas spp. preferably Pseudomonas aeruginosa.
  • the target cell is a microbial cell selected from the group consisting of eukaryotic cells and prokaryotic cells.
  • the target cell is a prokaryotic cell, preferably a bacterial cell, preferably a Gram negative bacterial cell, preferably a Pseudomonas spp. cell, preferably a Pseudomonas aeruginosa cell.
  • the polynucleotide in association with the anti-microbial agent is complexed to the anti-microbial agent by at least one chemical bond.
  • the chemical bond is a coordination bond or an ionic bond.
  • the polynucleotide in association with the anti-microbial agent is, or comprises a nucleic acid sequence that encodes a polynucleotide scaffold. In one embodiment the polynucleotide in association with the anti-microbial agent, is or consists essentially of a nucleic acid sequence that encodes a polynucleotide scaffold. In one embodiment the polynucleotide in association with the anti-microbial agent consists of a nucleic acid sequence that encodes a polynucleotide scaffold.
  • the polynucleotide scaffold or at least a portion of the polynucleotide scaffold, comprises a nucleic acid sequence that associates and/or interacts with a targeting component as described herein, and contributes to the binding of a target cell or molecule by the targeting component.
  • the targeting component is an aptamer or a functional fragment thereof.
  • x is an integer that is 3, 4, 5, 6 or 7 and y is an integer that is 2, 3, 4 or 5.
  • the polynucleotide scaffold comprises at least one additional nucleoside residue either 5' or 3' or both, of the nucleic acid sequence that associates with the ant-microbial agent.
  • the polynucleotide scaffold, or at least part of the polynucleotide scaffold is conjugated to the 5' end of the targeting component. In one embodiment the polynucleotide scaffold, or at least part of the polynucleotide scaffold is conjugated to the 3' end of the targeting component. In one embodiment a polynucleotide scaffold, or at least part of a polynucleotide scaffold is conjugated to the 5' end of the targeting component and a polynucleotide scaffold, or at least part of a polynucleotide scaffold, is conjugated to the 3' end of the targeting component. In one embodiment the polynucleotide scaffold is complexed with at least one atom of silver (Ag).
  • the at least one atom of Ag is complexed with the polynucleotide scaffold by at least one coordination bond.
  • the polynucleotide scaffold is complexed with a silver nanocluster (AgNC).
  • the AgNC is complexed with the polynucleotide scaffold by at least one coordination bond.
  • a silver nanocluster comprises at least 3 silver atoms (Ag). In one embodiment a silver nanocluster comprises about 3 to 50 silver atoms, preferably 3 to 50 silver atoms. In some embodiments, a silver nanocluster comprises about 3 to about 12 atoms, preferably 3 to about 12 atoms, preferably about 3 to 12 atoms, preferably 3 to at least 12 atoms, preferably at least 3 to 12 atoms, preferably 3 to 12 atoms.
  • polynucleotide scaffold comprises at least 4 nucleotides. In one embodiment the polynucleotide scaffold comprises at least 4 and less than 30 nucleotides. In one embodiment the polynucleotide scaffold comprises 4 to 30 nucleotides, preferably 4 to 29, preferably 4 to 28, preferably 4 to 27, preferably 4 to
  • (C x N y ) in the polynucleotide scaffold is at least 4 nucleotides. In one embodiment (C x N y ) in the polynucleotide scaffold comprises at least 4 and less than 30 nucleotides. In one embodiment (C x N y ) in the polynucleotide scaffold comprises 4 to 30 nucleotides, preferably 4 to 29, preferably 4 to 28, preferably 4 to
  • the polynucleotide scaffold consists essentially of at least 4 nucleotides. In one embodiment the polynucleotide scaffold consists essentially of at least 4 and less than 30 nucleotides. In one embodiment the polynucleotide scaffold consists essentially of 4 to 30 nucleotides, preferably 4 to 29, preferably 4 to 28, preferably 4 to 27, preferably 4 to 26, preferably 4 to 25 nucleotides.
  • (C x N y ) in the polynucleotide scaffold consists essentially of at least 4 and less than 30 nucleotides. In one embodiment (CxN y ) in the polynucleotide scaffold consists essentially of 4 to 30 nucleotides, preferably 4 to 29, preferably 4 to
  • (CxN y ) in the polynucleotide scaffold consists of 4 and less than 30 nucleotides. In one embodiment (C x N y ) in the polynucleotide scaffold consists of 4 to 30 nucleotides, preferably 4 to 29, preferably 4 to 28, preferably 4 to 27, preferably 4 to 26, preferably 4 to 25 nucleotides.
  • the DNA scaffold consists of at least 4 nucleotides. In one embodiment the DNA scaffold consists of at least 4 and less than 30 nucleotides. In one embodiment the DNA scaffold consists of 4 to 30 nucleotides, preferably 4 to 29, preferably 4 to 28, preferably 4 to 27, preferably 4 to 26, preferably 4 to 25 nucleotides.
  • the polynucleotide comprises about 10 nucleotides, preferably consists essentially of about 10 nucleotides, preferably consists of about 10 nucleotides. In one embodiment the polynucleotide comprises 10 nucleotides, preferably consists essentially of 10 nucleotides, preferably consists of 10 nucleotides.
  • polynucleotide scaffold comprises SEQ ID NO: 31 or a functional fragment thereof. In one embodiment the polynucleotide scaffold consists essentially of SEQ ID NO: 31 or a functional fragment thereof. In one embodiment the polynucleotide scaffold consists of SEQ ID NO: 31 or a functional fragment thereof. i- motif
  • C-rich sequences can fold to form i-motifs that are four stranded structures comprising two parallel duplexes hydrogen bonded in an antiparallel orientation by interchelated cytosine to hemiprotonated cytosine base pairs [29], in which the strands may originate from a single or multiple molecules [30, 34].
  • i-motifs can also form at neutral pH particularly when cytosine to cytosine base pairing is stabilized by Ag + ions [21]. Indeed, the reduction of Ag + stabilized i-motif to a fluorescent nanocluster has been employed as the basis of a sensitive assay for the detection of Ag + [35].
  • the inventors believe that the C-rich regions within certain aptamer sequences are non-suited to form intra-strand i-motifs as they do not contain a suitable loop region to allow i-motif folding.
  • modified aptamers containing a C-rich tract on both the 5' and 3' end of the DNA sequence can form an intra-strand i-motif by coming together with the addition of Ag + .
  • the inventors believe that the polynucleotide scaffolds described herein, alone or combined with aptamers as described herein, will form dimer or tetramer inter-strand i-motifs stabilized by Ag + , with two or four separate sequences coming together.
  • the CD spectra presented in example 3 are consistent with the inventor's
  • i-motif structures can be formed from polynucleotide sequences in many different ways, such as by varying the C-tract region nucleotides and length, as well as the nucleotides and lengths composing the bend in the i-motif structure.
  • any sequence that forms an i-motif at neutral and physiological pH due to the stabilising binding of Ag + within the i-motif structure will have antimicrobial activity, particularly bacteriostatic and/or bactericidal activity.
  • the inventors have also determined that the formation of a silver stabilised i-motif enhances the antimicrobial activity of silver ions in an anti-microbial component as described herein, particularly as compared with silver ions alone. While the exact mechanism is unknown, the inventors believe that the imotif structure formed by a polynucleotide as described herein acts to facilitate the uptake of an anti-microbial component as described herein, by the bacteria. This increased uptake results in enhanced inhibition of the growth and/or proliferation of both Gram-positive and Gram-negative bacterial species, particularly a) Gram-negative bacterial species, preferably Pseudomonas spp., preferably P. aeruginosa; Salmonella spp., preferably S.
  • enterica enterica
  • Klebsiella spp. preferably K. pneumoniae
  • Enterobacter spp. preferably E. cloacae
  • Acinetobacter spp. preferably A. baumannii and Escherichia coli and b) Gram-positive bacterial species, preferably Listeria spp., preferably L. innocua
  • Staphylococcus spp. preferably S. aureus or S. epididymis.
  • Circular dichroism is a simple spectroscopy method for detecting the formation of i- motif structures in DNA and is the most accepted method for detecting i-motif formation in the literature.
  • Other possible methods for i-motif detection include the interchelation of dyes and thermal difference spectroscopy. It was reasoned that the C-rich regions conjugated to the aptamers were not suited to form intra-strand i-motifs as they do not contain a suitable turn region to allow the strand to fold back on itself and form the i-motif structure and do not contain the four poly C regions required. However, without wishing to be bound by theory, the inventors believe there is the possibility that the aptamers containing a C-rich tract on both the 5' and 3' end of the DNA sequence.
  • the stem region of the aptamer brings the 5' and 3' poly C tracts into juxtaposition being functionally equivalent to a turn region in a full iMotif.
  • This 'split' half i-motif can form an intra-strand full i-motif upon the addition of Ag + by dimerising, the resulting structure being a full iMotif formed by two strands containing two poly C regions separated by alternative turn structure.
  • the possibility of dimer and tetramer inter-strand i-motifs stabilised by Ag + are also possible with two or four separate C-rich sequences coming together.
  • polynucleotide structures that form i-motifs by themselves are useful, when combined with an anti-microbial agent, as an anti-microbial therapeutic, either targeted to a particular cell or molecule with an aptamer or untargeted.
  • i-motif anti-microbial therapeutics as described herein are envisaged as useful for topical treatment of infections such as otitis externa, burn wounds and ulcers as well as treatment of infections in the cystic fibrosis lung, or in urinary tract.
  • i-motifs alone exhibit strong antimicrobial activity against a wide range of different bacterial species, supporting the utility of i- motifs as useful in broad spectrum antibiotic therapies.
  • i- motifs may be provided in the form of a solution, emulsion, bound to carrier nanoparticles or ai hydrogel for topical application or for use as a wash or lavage.
  • a solution comprising an anti-microbial agent that was an i-motif alone as described herein could be formulated at concentration of from 10 ⁇ to 0.0001 ⁇ active agent, depending on the application and addition of additives.
  • the action of i-motifs alone can be enhanced by treating simultaneously with EDTA, a common wash in topical treatments before application of a drug.
  • the inventors have unexpectedly found that the action of Ag + stabilised i-motifs is strongly synergistic when imotifs alone are combined with EDTA for co-treatment. This synergistic action is seen against a large number of diverse Gram positive and negative bacterial species.
  • the invention relates to an anti-microbial component (AMC) comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or a polynucleotide structure that chelates at least one metal ion, or both, and ii) an anti-microbial agent.
  • AMC anti-microbial component
  • an anti-microbial component comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or a polynucleotide structure that chelates at least one metal ion, or both, and ii) an anti-microbial agent to inhibit the growth and/or proliferation of at least one bacterial species.
  • i) is a polynucleotide structure that chelates at least one metal ion.
  • the anti-microbial component comprises the polynucleotide structure in association with the anti-microbial agent, wherein the polynucleotide structure comprises an i-motif structure and the anti-microbial agent is at least one metal ion.
  • the antimicrobial component consists essentially of, preferably consists of, the polynucleotide structure and the at least one metal ion.
  • the polynucleotide structure comprises, consists essentially of, or consists of a single nucleic acid strand that forms the i-motif structure.
  • the polynucleotide structure comprises, consists essentially of, or consists of at least two, preferably four, nucleic acid strands that form the imotif structure.
  • the imotif structure is formed under suitable conditions.
  • the suitable conditions are conditions in which the nucleic acid strand or strands that form the imotif structure exist in free solution at neutral pH in a configuration that is not a full formed i-motif, wherein the addition of the at least one metal ion stabilizes the nucleic acid strand or strands to form the i-motif structure.
  • the at least one metal ion is Cu 2+ or Ag + , preferably Ag + .
  • nucleic acid strand or strands that form the imotif structure comprise a nucleic acid sequence comprising at least one, preferably at least two, preferably four poly C tracts (pCts).
  • nucleic acid strand or strands that form the imotif structure comprise a nucleic acid sequence comprising at least one pair of pCts separated by a turn region (TR), and have the formula (pCt-TR-pCt)n, wherein n is an integer from 1 to at least 10, preferably to at least 100, preferably to at least 1000, preferably to at least 10,000, preferably to at least 100,000.
  • the turn regions compise only A and/or T nucleotide residues.
  • the turn regions are selected from the group consisting of A, T, AA, TT, AT, TA, AAT, ATA, TAA, ATT, TTA, TTT, AAAA, AAAT, AATA, ATAA, TAAA, AATT, ATTA, TTAA, ATTT, TTTA, and ⁇ .
  • the turn region is a targeting component as described herein. In one embodiment the turn region is an aptamer as described herein.
  • poly-C tracts or turn regions or both are variant poly-C tracts or variant turn regions, wherein a variant poly-C tract or variant turn region differs from a poly-C tract or turn region by the inclusion of either a single G residue, or a doublet GG residue pair.
  • Non-limiting examples of such nucleic acid strands having variant poly-C tracts and/or variant turn regions are provided in SEQ ID NO: 38-SEQ ID NO: 43.
  • the polynucleotide structure is formed from a nucleic acid strand comprising a concatamer of nucleic acid sequences having alternating pCts and TRs. In one embodiment the polynucleotide structure is formed from four separate nucleic acid strands each strand comprising at least one, preferably one, pCt.
  • the i-motif structure consists essentially of, preferably consists of, a single nucleic acid strand, or of at least two separate nucleic acid strands, or of four separate nucleic acid strands.
  • x is an integer that is 3, 4, 5, 6 or 7 and y is an integer that is 2, 3, 4 or 5.
  • the nucleic acid sequence that forms the i-motif, or at least part of the i-motif comprises at least one additional nucleoside residue 5' or 3' of the nucleic acid sequence that forms the i-motif, or that forms part of the i-motif.
  • the polynucleotide structure comprises, preferably consists essentially of, preferably consists of, a nucleic acid selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43.
  • the at least one bacterial species is a Gram-negative bacterial species.
  • the Gram-negative bacterial species is a mammalian pathogen, preferably a human, horse or dog pathogen.
  • the Gram-negative bacterial species is selected from the group consisting of Pseudomonas spp. belonging to the P. aeruginosa group, P. chlolraphis group, P. fluorescens group, P. pertucinogena group, P. putida group, P. stutzeri group, or the P. syringae group, preferably wherein the Pseudomonas spp. is P. aeruginosa, P. florescens, P. putida, P. stutzeri, P. tabaci, P. angulata, P. phaseolicola, P. pisi, P. glycinea, P.
  • solanacearum P. caryophylli, P. cepacia, P. marginalis, P. savastonoi, P. marginata or P. syringae; Salmonella spp., preferably S. enterica; Klebsiella spp., preferably K. pneumoniae; Enterobacter spp., preferably E. cloacae; Acinetobacter spp., preferably A. baumannii and Escherichia coll.
  • the least one bacterial species is a Gram-positive bacterial species.
  • the Gram-positive bacterial species is a mammalian pathogen, preferably a human, horse or dog pathogen.
  • the Gram-positive bacterial species is a Listeria spp., preferably L. innocua; and/or a Staphylococcus spp., preferably S. aureus or S. epididymis.
  • the invention relates to the use of an anti-microbial component to inhibit the growth and/or proliferation of at least one bacterial species, wherein the anti-microbial component consists essentially of a polynucleotide structure in association with an anti-microbial agent, wherein the polynucleotide structure comprises an i-motif structure, wherein the anti-microbial agent is at least one metal ion, preferably at least one Ag + , wherein the imotif structure comprises a single nucleic acid strand that forms the i- motif structure or at least two, preferably four, nucleic acid strands that form the imotif structure, wherein the nucleic acid strand or strands that form the i-motif structure comprise a nucleic acid sequence compris
  • an anti-microbial component comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or a polynucleotide structure that chelates at least one metal ion, or both, and an anti-microbial agent in the manufacture of a medicament for treating microbial infection, wherein the antimicrobial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion.
  • the invention in another aspect relates to a method of inhibiting the growth and/or proliferation of at least one bacterial species comprising contacting the at least one bacterial species with an anti-microbial component comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or a polynucleotide structure that chelates at least one metal ion, or both, and ii) an anti-microbial agent, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an i-motif structure and the anti-microbial agent is at least one metal ion.
  • an anti-microbial component comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or a polynucleotide structure that chelates at least one metal ion, or both, and ii) an anti-microbial agent, where
  • the invention in another aspect relates to a method of treating a microbial infection comprising administering an anti-microbial component comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or a polynucleotide that chelates at least one metal ion, or both, and an anti-microbial agent to a subject in need thereof, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion.
  • an anti-microbial component comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or a polynucleotide structure that chelates at least one metal ion, or both, and ii) an anti-microbial agent for use in inhibiting the growth and/or proliferation of at least one bacterial species treating, or when used to treat, microbial infection, wherein the polynucleotide in i) comprises an i-motif structure and the anti-microbial agent in ii) is at least one metal ion.
  • any of the aspects set out herein including aspects that relate to and/or encompass imotif structures and aspects that relate to and/or encompass antimicrobial combinations of formulas I, II and Ila, and including uses and methods of the anti-microbial components and anti-microbial combinations disclosed herein.
  • a polynucleotide structure as described herein either forms, or forms part of an "i-motif".
  • the polynucleotide in association with the anti-microbial agent comprises a polynucleotide scaffold.
  • the polynucleotide scaffold comprises a polynucleotide that forms, or that forms part of, an i-motif.
  • the polynucleotide forms, or forms part of, an i-motif that binds at least one metal ion.
  • the at least one metal ion is selected from the group consisting of silver ions and copper ions, preferably silver ions.
  • the polynucleotide that chelates at least one metal ion a nucleic acid sequence that forms, or that forms part of, an i-motif.
  • the polynucleotide forms, or forms part of, an i-motif that binds at least one metal ion.
  • the at least one metal ion is selected from the group consisting of silver ions and copper ions, preferably silver ions.
  • a polynucleotide that forms an i-motif comprises a nucleic acid sequence having at least two repeating poly C tracts separating a turn region with the i-motif being formed by concatermerisation of the nucleic acid having the alternating poly C tracts separating the turn region.
  • the polynucleotide that forms an i-motif, or that forms part of an i-motif is conjugated to the 5' end of a targeting component. In one embodiment the polynucleotide that forms an i-motif, or that forms part of an i-motif is conjugated to the 3' end of a targeting component. In one embodiment a polynucleotide that forms an i-motif, or that forms part of an i-motif is conjugated to the 5' end of a targeting component and a polynucleotide that forms an i-motif, or that forms part of an i-motif is conjugated to the 3' end of a targeting component.
  • the polynucleotide that forms an i-motif, or that forms part of an i-motif comprises about 24 nucleotides, preferably consists essentially of about 24 nucleotides, preferably consists of about 24 nucleotides. In one embodiment the polynucleotide that forms an i-motif, or that forms part of an i-motif comprises 24 nucleotides, preferably consists essentially of 24 nucleotides, preferably consists of 24 nucleotides.
  • the polynucleotide that forms an i-motif, or that forms part of an i-motif comprises a nucleic acid selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 41 and SEQ ID NO: 43, or a functional fragment thereof.
  • the nucleic acid selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 41 and SEQ ID NO: 43, or a functional fragment thereof.
  • polynucleotide that forms an i-motif, or that forms part of an i-motif consists essentially of a nucleic acid selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 41 and SEQ ID NO: 43, or a functional fragment thereof.
  • the polynucleotide that forms an i-motif, or that forms part of an i-motif consists of a nucleic acid selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 41 and SEQ ID NO: 43, or a functional fragment thereof.
  • the anti-microbial agent is selected from the group consisting of anti-viral agents, anti-bacterial agents, anti-protist agents and anti-fungal agents.
  • the anti-microbial agent is an anti-bacterial agent.
  • the anti-microbial agent is an organic or inorganic anti-microbial agent. In one embodiment the organic anti-microbial agent is an antibiotic.
  • the antibiotic is selected from the group consisting of penicillins,
  • cephalosporins cephalosporins, tetracyclins, macrolides, glycopeptides, quinolones and
  • the inorganic anti-microbial agent is selected from the group consisting of metals and small molecules.
  • the metal is a noble metal.
  • the metal is elemental metal or is a metal ion.
  • the elemental metal is silver (Ag) or copper (Cu), preferably silver.
  • the elemental metal is Ag.
  • the Ag is selected from the group consisting of at least one atom of Ag, a complex of at least 2 atoms of Ag, an Ag nanocluster, and a Ag nanoparticle.
  • the Ag is comprised in an Ag nanocluster (AgNC).
  • the Ag nanocluster comprises about 3 to 50 Ag atoms, preferably 3 to 50 atoms of Ag.
  • an Ag nanocluster comprises about 3 to about 12 atoms, preferably 3 to about 12 atoms, preferably about 3 to 12 atoms, preferably 3 to at least 12 atoms, preferably at least 3 to 12 atoms, preferably 3 to 12 atoms of Ag.
  • the metal ion is Ag + .
  • the anti-microbial agent is Ag, a nanocluster of Ag, a nanoparticle of Ag, or Ag + .
  • the anti-microbial agent is Cu, a nanocluster of Cu, a nanoparticle of Cu, Cu + or Cu ++ .
  • the invention relates to a combination of formula I:
  • TC a targeting component that specifically binds to a target molecule or cell
  • AMC an anti-microbial component comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or is a polynucleotide that chelates at least one metal ion, or both, and ii) an anti-microbial agent.
  • targeting and/or anti-microbial components and/or agents are also specifically contemplated herein as part of this aspect of the invention that relates to the combination of formula II, and are specifically contemplated herein.
  • AptPA is an aptamer or functional fragment thereof that specifically binds a Pseudomonas aeruginosa cell
  • targeting and/or anti-microbial components and/or agents are also specifically contemplated herein as part of this aspect of the invention that relates to the combination of formula Ila, and are specifically contemplated herein.
  • the polynucleotide in i) in formula II and/or formula Ila comprises a nucleic acid sequence having at least 50% nucleic acid sequence identity with SEQ ID NO: 31 and SEQ ID NO: 32 or with any one of SEQ ID NO: 38 to SEQ ID NO: 43.
  • the polynucleotide in i) comprises a nucleic acid sequence having at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% nucleic acid sequence identity with SEQ ID NO: 31 or SEQ ID NO: 32 or with any one of SEQ ID NO: 38 to SEQ ID NO: 43.
  • polynucleotide in i) consists essentially of SEQ ID NO: 31 or SEQ ID NO: 32 or with any one of SEQ ID NO: 38 to SEQ ID NO: 43. In one embodiment the polynucleotide in i) consists of SEQ ID NO: 31 or SEQ ID NO: 32 or with any one of SEQ ID NO: 38 to SEQ ID NO: 43.
  • AptPA is an aptamer that specifically binds a Pseudomonas aeruginosa cell as described herein for the first, or any other aspect of the invention.
  • the anti- microbial component, polynucleotide in i), and the anti-microbial agent are as described herein for the first or any other aspect of the invention.
  • the invention in another aspect relates to a method of making an anti-microbial combination comprising : a) synthesizing a polynucleotide comprising a targeting component comprising a polynucleotide that specifically binds to a target molecule or cell, and an antimicrobial component comprising a nucleic acid sequence that forms a metal nanocluster with at least three metal atoms, or is a nucleic acid sequence that chelates at least one metal ion, or both, and b) associating the polynucleotide synthesized in a) with at least one antimicrobial agent to make the anti-microbial combination.
  • the method comprises an initial step of designing the targeting component comprising making a polynucleotide that specifically binds to a target molecule or cell.
  • making the polynucleotide comprises evolving the polynucleotide using SELEX.
  • making the polynucleotide comprises amplifying at nucleic acid template using SEQ ID NO: 33 and SEQ ID NO: 34 as amplification primers.
  • the method comprises an initial step of designing the anti- microbial component.
  • designing the anti-microbial component comprises designing a nucleic acid sequence that forms a metal nanocluster with at least three metal atoms, or is a nucleic acid sequence that chelates at least one metal ion, or both.
  • the metal nanocluster is a silver or copper nanocluster.
  • the metal ion is a silver or copper ion.
  • the method comprises the step of confirming that the targeting component specifically binds to a target molecule or cell.
  • associating the polynucleotide synthesized in a) comprises binding at least 3 silver or copper atoms to the polynucleotide.
  • binding comprises binding at least 3 silver or copper ions to the polynucleotide to form a complex, and then contacting the complex with at least one reducing agent to form a silver (AgNC) or copper (CuNC) nanocluster.
  • the invention in another aspect relates to a composition
  • a composition comprising an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, or a combination of formula II or Ila as described herein for any aspect of the invention and a carrier, diluent or excipient.
  • composition is a pharmaceutical composition.
  • composition or pharmaceutical composition comprises an effective amount of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila.
  • effective amount is a therapeutically effective amount.
  • the effective amount is an amount of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila that kills at least 50%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, at least 99.99%, at least 99.995%, or preferably at least 99.999%of a target microorganism, preferably a target bacteria, as described herein for any aspect of the invention, when contacted to the target microorganism.
  • the target microorganism comprises a starting population of cells (i.e., the number of cells before treatment begins) of at least 1.0 x 10 5 cells.
  • microorganism preferably the target bacteria, occurs in less than about 4 hours after the target microorganism is contacted . Preferably killing occurs in less than 4 hours.
  • killing occurs in less than about 3 hours, preferably less than about 2 hours, preferably less than about 1 hour. In one embodiment killing occurs in less than 3 hours, preferably less than 2 hours, preferably less than 1 hour.
  • concentration of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila in the composition or pharmaceutical composition is from about 500 nM to about 10 ⁇ , or from about 500 nM to 10 ⁇ , or from 500 nM to about 10 ⁇ , or from 500 nM to 10 ⁇ . In one embodiment the concentration is about 1 ⁇ to about 5 ⁇ , or is about 1 ⁇ to 5 ⁇ , or is 1 ⁇ to about 5 ⁇ or is 1 u ⁇ to 5 ⁇ .
  • the concentration is less than 5 ⁇ , about 5 ⁇ or is 5 ⁇ . In one embodiment where the antimicrobial agent is Ag + , Cu + or Cu ++ as described herein, the concentration is less than 1 ⁇ , is about 1 ⁇ , or is 1 ⁇ .
  • the concentration of the combination of formula I or II or Ila or of the anti-microbial component comprising an i-motif and silver ions shows therapeutic activity against a target cell with an IC50 of less than 10 ⁇ , preferably less than 7.5 ⁇ , preferably less than 5 ⁇ , preferably less than 2.5 ⁇ , preferably less than 1 ⁇ . In one embodiment the concentration of the combination of formula I or II or Ila shows therapeutic activity against a target cell with an IC50 of about 7.5 ⁇ , preferably about 5 ⁇ , preferably about 2.5 ⁇ , preferably about 1 ⁇ .
  • the carrier, diluent or excipient is a buffer. In one embodiment the buffer is a zwitterionic buffer.
  • the zwitterionic buffer is selected from the group consisting of MES, MOPS, HEPES and TRIS, preferably MES.
  • the buffer is an inorganic buffer.
  • the inorganic buffer is selected from the group consisting of citrate, acetate, phosphate and cacodylate. Buffers with low concentrations of chloride ions are preferred to prevent precipitation of AgCI.
  • the buffer maintains the composition or pharmaceutical composition in a pH range of about 6 to about 8 or of about 6 to 8 or of 6 to about 8 or of 6 to 8, preferably about 6.5 to about 7.5 or about 6.5 to 7.5 or 6.5 to about 7.5 or 6.5 to 7.5, preferably about pH 6.5, 7 or 7.5, preferably at pH 6.5 ⁇ .2, 7 ⁇ .2 or 7.5 ⁇ .2, preferably at pH 6.5, 7 or 7.5, preferably at pH 6.5.
  • composition or pharmaceutical composition comprises pharmaceutically acceptable carriers, proteins, small peptides, salts, excipients, thickeners, diluents, buffers, preservatives, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers and the like in addition to an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, or a combination of formula II or Ila as described herein.
  • pharmaceutically acceptable carriers proteins, small peptides, salts, excipients, thickeners, diluents, buffers, preservatives, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers and the like in addition to an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, or a combination of formula II or Ila as described herein.
  • compositions and formulations can be used as described herein.
  • a pharmaceutically acceptable carrier may be liquid or solid and is selected as known in the art, in view of a planned manner of administration.
  • a pharmaceutically acceptable carrier provides for the desired bulk, consistency, or other
  • a pharmaceutically acceptable carrier typically includes binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone (PVP) or hydroxypropyl
  • binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone (PVP) or hydroxypropyl
  • methylcellulose and the like, fillers such as lactose or other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrates (e.g., starch, sodium starch glycolate, etc.); or wetting agents (e.g., sodium lauryl sulphate, etc.).
  • the carrier is formulated to allow application of a composition or pharmaceutical composition as described herein by aerosol directly to the lungs.
  • Penetration enhancers may be included in pharmaceutical compositions in order to enhance the delivery of a useful agent.
  • penetration enhancers include fatty acids, bile salts, chelating agents, surfactants and non-surfactants, but are not limited thereto.
  • Single penetration enhancers may be used alone or in combination with any other penetration enhancer disclosed herein.
  • fatty acids (and derivatives thereof) useful as penetration enhancers include, but are not limited to, oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid and physiologically acceptable salts thereof.
  • Suitable chelating agents are well known and disclosed in the art and will include a number of different agents that may be selected by the skilled worker, the skilled worker being aware that the selected agent should not introduce chloride ions into the composition.
  • agents include, but are not limited to, disodium
  • EDTA ethylenediaminetetraacetate
  • citric acid citric acid
  • salicylates e.g., sodium salicylate, 5- methoxysalicylate and homovanilate
  • N-acyl derivatives of collagen e.g., N-acyl derivatives of collagen.
  • surfactants are well known and disclosed in the art. Examples include, but are not limited to, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. Examples of non-surfactants include, but are not limited to, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives and non-steroidal antiinflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone
  • the anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, or a combination of formula II as described herein can be formulated in pharmaceutical compositions that contain additional functional or therapeutic components.
  • Such other components can be considered adjunct components as may be conventionally found in pharmaceutical compositions, at their art-established usage levels.
  • Such components include compatible pharmaceutically-active materials such as local anaesthetics or anti-inflammatory agents.
  • Additional materials useful in physically formulating various dosage forms of a pharmaceutical composition may also be included, such as dyes, flavouring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • a person skilled in the art will be able to formulate a anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, or a combination of formula II or Ila as described herein as a composition, preferably a pharmaceutical composition, by determining an appropriate mode of administration of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila with reference to the literature and as described herein, and then formulating the composition for such mode with reference to the literature and as described herein.
  • a formulation of the composition for topical application would be preferred for the treatment and prevention of certain microbial infections of the skin or mucosa
  • a formulation of the composition for systemic application would be preferred for the treatment of systemic microbial infections or localized internal microbial infections, and aerosol delivery to the lungs, such as when treating cystic fibrosis, but not limited thereto.
  • the pharmaceutical composition is formulated for administration, or is in a form for administration, to a subject in need thereof.
  • administration is selected from the group consisting of is topical, intranasal, epidermal, transdermal, oral or parenteral.
  • parenteral administration is selected from the group consisting of direct application, systemic, subcutaneous, intraperitoneal or intramuscular injection, intravenous drip or infusion, inhalation, insufflation or intrathecal or intraventricular administration. In one embodiment administration is by aerosol delivery.
  • the anti-microbial combination, targeting component, antimicrobial component, combination of formula I, or combination of formula II or Ila as described herein is formulated for, or is in a form for, parenteral administration in any appropriate solution, including sterile aqueous solutions which may also contain buffers, diluents and other suitable additives
  • the anti-microbial combination, targeting component, anti- microbial component, combination of formula I, or combination of formula II or Ila as described herein is formulated for, or is in a form for oral administration in powders or granules, aqueous or non-aqueous suspensions or solutions, capsules, pills, lozenges or tablets. Thickeners, flavouring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • the anti-microbial combination, targeting component, antimicrobial component, combination of formula I, or combination of formula II or Ila as described herein is formulated for, or is in a form for topical, aerosol, or direct administration in transdermal patches, subdermal implants, ointments, lotions, creams, gels, drops, pastes, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • the direct administration is direct application or local application.
  • direct or local application comprises application of the antimicrobial combination, targeting component, anti-microbial component, combination of formula I or combination of formula II or Ila as described herein in combination with a delivery reagent or additional anti-microbial agent, but not limited thereto.
  • an anti-microbial combination a targeting component, an antimicrobial component, a combination of formula I, or a combination of formula II or Ila as described herein with reference to the literature and as described herein.
  • a systemic application would be preferred for the treatment and prevention of certain microbial infections whereas a local application would be preferred for the treatment of others, but not limited thereto.
  • composition or pharmaceutical composition comprises at least one formulation agent. In one embodiment, the composition comprises at least two formulation agents. In one embodiment the composition or pharmaceutical composition further comprises an additional anti-microbial agent. In one embodiment the composition or pharmaceutical composition comprises a combination of one or more additional anti-microbial agents and one or more formulation agents. In some embodiments the composition or pharmaceutical composition is formulated as pre-prepared composition or in a concentrated form. In one embodiment the additional anti-microbial agent is selected from the group consisting of anti-viral agents, anti-bacterial agents, anti-protist agents and antifungal agents.
  • an additional anti-microbial agent is an agent that is capable of contributing to the control (e.g., treatment and/or prevention) of microbial infection, particularly bacterial infection, preferably infection by Pseudomonas spp. bacteria, more preferably infection by P. aeruginosa, but not limited thereto.
  • Suitable additional anti-microbial agents for use in the present invention may be capable of controlling Pseudomonas spp., particularly P. aeruginosa directly, or may be capable of potentiating the effect of any one or all of SEQ ID NO: 1 to 30 and 38 to 43 for controlling Pseudomonas spp., particularly P. aeruginosa.
  • Additional antimicrobial agents may be included directly in the composition of or useful in the invention, or may be administered separately, either simultaneously or sequentially as appropriate according to a use or method of the invention.
  • the additional anti-microbial agent is an organic or inorganic anti- microbial agent.
  • the organic anti-microbial agent is an organic anti-microbial agent.
  • the antibacterial agent is an antibiotic.
  • the antibiotic is selected from the group consisting of penicillins, cephalosporins, tetracyclins, macrolides, glycopeptides, quinolones and
  • the inorganic additional anti-microbial agent is selected from the group consisting of metals and small molecules.
  • the metal is a noble metal.
  • the metal is elemental metal or is a metal ion.
  • the elemental metal or metal ion is the elemental form of, or an ion of, silver (Ag), mercury (Hg), platinum (Pt), gold (Au), copper (Cu) or arsenic (As).
  • the invention relates to the use of an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, a combination of formula II or Ila, or a composition as described herein for the first or any other aspect of the invention, in the manufacture of a medicament for treating microbial infection.
  • the medicament comprises an effective amount of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, combination of formula II or Ila, or
  • the effective amount is a therapeutically effective amount.
  • the medicament comprises at least one additional anti-microbial agent.
  • the at least one additional anti-microbial agent is an antibiotic.
  • the medicament comprises an effective amount of the additional anti-microbial agent. In one embodiment the effective amount of the at least one additional anti-microbial agent is a therapeutically effective amount.
  • the medicament consists essentially of an effective amount of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila and an additional antimicrobial agent.
  • the effective amount of the additional anti- microbial agent is a therapeutically effective amount.
  • the medicament is formulated for administration, or is in a form for administration, to a subject in need thereof.
  • the medicament is in a form for, or is formulated for topical, intranasal, epidermal, transdermal, oral or parenteral administration.
  • parenteral administration is selected from the group consisting of direct application, systemic, subcutaneous, intraperitoneal or intramuscular injection, intravenous drip or infusion, inhalation, insufflation or intrathecal or intraventricular administration.
  • the medicament is in a form for, or is formulated for, parenteral administration in any appropriate solution, preferably in a sterile aqueous solution which may also contain buffers, diluents and other suitable additives.
  • administration selected from the group consisting of a powder, a granule, an aqueous suspension, an aqueous solution, a non-aqueous suspension, a non-aqueous solution, a capsule, a pill, a lozenge, and a tablet.
  • the addition of one or more of the following may be desirable: thickeners, flavouring agents, diluents, emulsifiers, dispersing aids or binders.
  • the medicament is formulated for, or is in a form for topical or direct administration selected from the group consisting of transdermal patches, subdermal implants, ointments, lotions, creams, gels, drops, pastes, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be used as required or desired in this embodiment.
  • the direct administration is direct application or local application.
  • direct or local application comprises application of the medicament in combination with a delivery reagent or additional anti-microbial agent.
  • a person skilled in the art will be able to choose the appropriate mode of
  • the medicament is for, is formulated for, or is in a form for administration separately, simultaneously or sequentially with an additional antimicrobial agent.
  • one additional anti-microbial agent that may be included in the composition of, or for use in the invention is an antibiotic that is, or is suspected of being effective against a target cell, particularly a target bacterial cell.
  • the target bacterial cell is a Gram negative bacterial cell.
  • the inventors have determined that various combinations of a targeting component, anti-microbial component, combination of formula I, combination of formula II or Ila, or composition as described herein and an antibiotic, particularly ciprofloxin, have an interworking relationship and provide an unexpected synergistic bactericidal effect when used together as disclosed herein.
  • the medicament comprises an anti-microbial combination, targeting component, anti-microbial component, combination of formula I,
  • the medicament comprises an anti-microbial combination, targeting component, anti-microbial component, combination of formula I,
  • the invention relates to the use of an anti-microbial combination, targeting component, anti-microbial component, combination of formula I,
  • the invention relates to the use of an anti-microbial component as described herein in the manufacture of a medicament for treating microbial infection, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an i-motif structure and the anti-microbial agent is at least one metal ion.
  • targeting and/or anti-microbial components and/or agents are also specifically contemplated herein as part of this aspect of the invention that relates to the use of an anti-microbial component as described herein in the manufacture of a medicament for treating microbial infection, and are specifically contemplated herein.
  • the invention relates to use of an anti-microbial component as described herein to inhibit the growth and/or proliferation of at least one bacterial species, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an i- motif structure and the anti-microbial agent is at least one metal ion.
  • the anti-microbial component inhibits the growth and/or proliferation of at least one bacterial species.
  • the antibacterial component is bacteriostatic or bactericidal or both for the at least one bacterial species.
  • the at least one bacterial species is a Gram-negative bacterial species.
  • the Gram-negative bacterial species is selected from the group consisting of Pseudomonas spp. belonging to the P. aeruginosa group, P. chlolraphis group, P. fluorescens group, P. pertucinogena group, P. putida group, P. stutzeri group, or the P. syringae group, preferably wherein the Pseudomonas spp. is P. aeruginosa, P. florescens, P. putida, P. stutzeri, P. tabaci, P. angulata, P. phaseolicola, P. pisi, P. glycinea, P.
  • solanacearum P. caryophylli, P. cepacia, P. marginalis, P. savastonoi, P. marginata or P. syringae- Salmonella spp., preferably S. enterica; Klebsiella spp., preferably K. pneumoniae; Enterobacter spp., preferably E. cloacae; Acinetobacter spp., preferably A. baumannii and Escherichia coll.
  • the Gram-negative bacterial species is selected from the group consisting of Xanthomonas spp., preferably X. phaseoli, X. oryzae, X. runi, X. juglandis, X. campestris or X. vascularum; Erwinia spp., preferably E. amylovora, E. tracheiphila, E. stewartii or E. carotovora; Corynebacterium spp., preferably C.
  • the at least one bacterial species is a Gram-positive bacterial species.
  • the Gram-positive bacterial species is a Listeria spp., preferably L. innocua; and/or a Staphylococcus spp., preferably S. aureus or S. epididymis.
  • an anti-microbial component as described herein is in the form of, or is formulated as a disinfectant.
  • an anti-microbial component as described herein as a disinfectant is believed to be within the skill of those in the art in view of the present disclosure and common general knowledge.
  • the combination is formulated as, or is in the form of, a composition comprising an anti-microbial component as described herein and a carrier, diluent or excipient.
  • the composition consists essentially of the anti-microbial component.
  • the carrier, diluent or excipient is a buffer.
  • the buffer is a zwitterionic buffer.
  • the zwitterionic buffer is selected from the group consisting of MES, MOPS, HEPES and TRIS, preferably MES or MOPS.
  • the buffer is an inorganic buffer.
  • the inorganic buffer is selected from the group consisting of citrate, acetate, phosphate, carbonate and cacodylate. Buffers with low concentrations of chloride ions are preferred to prevent precipitation of AgCI.
  • the buffer maintains the composition in a pH range of about 6 to about 8 or of about 6 to 8 or of 6 to about 8 or of 6 to 8, preferably about 6.5 to about 7.5 or about 6.5 to 7.5 or 6.5 to about 7.5 or 6.5 to 7.5, preferably about pH 6.5, 7 or 7.5, preferably at pH 6.5 ⁇ .2, 7 ⁇ .2 or 7.5 ⁇ .2, preferably at pH 6.5, 7 or 7.5, preferably at pH 6.5.
  • an anti-microbial component as a composition in the form of a solid, liquid, paste, gel, particle, nanoparticle, emulsion, cream, ointment, lotion, liniment, solution, suspension, stick, block, pill, lozenge, powder, slurry, mist or vapour for use to inhibit the growth and/or proliferation of at least one Gram-negative bacterial species, or to treat a Gram-negative bacterial infection, disease and/or condition as described herein is believed to be within the skill of those in the art as described herein and in light of common general knowledge.
  • the anti-microbial component is formulated as a composition, pharmaceutical or cosmetic composition that comprises acceptable carriers, particularly pharmaceutically acceptable or cosmetically acceptable carriers, proteins, small peptides, salts, excipients, thickeners, diluents, buffers, preservatives, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and/or other carriers in addition to the anti-microbial component.
  • acceptable carriers particularly pharmaceutically acceptable or cosmetically acceptable carriers, proteins, small peptides, salts, excipients, thickeners, diluents, buffers, preservatives, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and/or other carriers in addition to the anti-microbial component.
  • an anti-microbial component as described herein as a composition, particularly a pharmaceutical or cosmetic composition, by determining an appropriate mode of use, application and/or administration of the composition with reference to the literature and as described herein, and then formulating the composition for such mode with reference to the literature and as described herein.
  • a formulation of the composition as a pharmaceutical composition for topical application would be preferred for inhibiting the growth and/or proliferation of certain Gram-negative bacteria, or for the treatment and prevention of certain Gram-negative bacterial infections, diseases and/or conditions of the skin or mucosa that are caused by and/or associated with at least one Gram-negative bacterial species.
  • a formulation of the composition as a pharmaceutical composition for systemic application would be preferred for the treatment of systemic or localized internal bacterial infections, diseases and/or conditions of the skin or mucosa that are caused by and/or associated with at least one Gram-negative bacterial species.
  • an anti-microbial component or pharmaceutical composition as contemplated herein may be formulated according to conventional pharmaceutical practice and may be: Semisolid formulations: Gels, pastes, mixtures. Liquid formulations: Solutions, suspensions, drenches, emulsions.
  • an anti-microbial component as described herein to inhibit the growth and/or proliferation of at least one bacterial species
  • the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an i motif structure and the anti-microbial agent is at least one metal ion, and are specifically contemplated herein.
  • the invention in another aspect relates to a method of inhibiting the growth and/or proliferation of at least one bacterial species comprising contacting the at least one bacterial species with an anti-microbial component as described herein, wherein the anti-microbial component comprises a polynucleotide in association with an antimicrobial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion.
  • contacting comprises contacting an object or part thereof that comprises the at least one bacterial species.
  • the at least one bacterial species is a Gram-negative bacterial species.
  • the at least one bacterial species is a Gram-positive bacterial species.
  • contacting comprises contacting a surface in and/or on the object or part thereof.
  • contacting is for a sufficient time to allow the combination or composition to inhibit the growth and/or proliferation of the at least one Gram- negative and/or Gram-positive bacterial species on and/or in the object.
  • applying is directly applying. In one embodiment applying is indirectly applying.
  • applying comprises applying the anti-microbial component to the object or part thereof at least two times.
  • applying is applying as a coating or partial coating.
  • applying comprises applying the combination or composition at least lx, or 2x, or 3x, or 4x, or 5x, or 6x, or 7x, or 8x, or 9x, preferably lOx, or more.
  • applying is least lx per day (lx/d), at least 2x/d, at least 3x/d, at least 4x/day, at least 5x/day, at least 6x/day, at least 7x/day, at least 8x/day, at least 9x/day, at least lOx/day.
  • the object is an animal or part thereof, or plant or part thereof.
  • the animal is a mammal.
  • the mammal is selected from the group consisting of canines, felines, bovines, ovines, equines, cervines, caprines, porcines, lagomorphs, rodents, camelids and hominids.
  • the mammal is selected from the group consisting of cats, dogs, rats, stoats, ferrets, possums, guinea pigs, mice, hamsters, zebra, elephants, lions, tigers, cheetah, monkeys, apes, macaques, tarsiers, lemurs, giraffes, prairie dogs, meerkats, bears, otters, tapiers, cows, horses, pigs, sheep, goats, deer, minks, hippopotami and humans.
  • the animal is a bird selected from the group consisting of chickens, ducks, pheasants, pigeons, ostriches, turkeys and geese.
  • the part of the animal is the hair, skin or hide, preferably human, cow, deer, sheep or horse hair, skin or hide.
  • the part of the plant is selected from the group consisting of roots, shoots, stalks, stems, trunks, branches, leaves, buds, flowers, and seeds.
  • contacting is to an animal or part thereof, and the at least one bacterial species is a Gram-negative bacterial species.
  • the Gram-negative bacterial species is selected from the group consisting of Pseudomonas spp. belonging to the P. aeruginosa group, P. chlolraphis group, P. fluorescens group, P. pertucinogena group, P. putida group, P. stutzeri group, or the P. syringae group, preferably wherein the Pseudomonas spp. is P.
  • savastonoi P. marginata or P. syringae- Salmonella spp., preferably S. enterica; Klebsiella spp., preferably K. pneumoniae; Enterobacter spp., preferably E. cloacae; Acinetobacter spp., preferably A. baumannii and Escherichia coll.
  • contacting is to an animal or part thereof, and the at least one bacterial species is a Gram-negative bacterial species.
  • the Gram-positive bacterial species is a Listeria spp., preferably L. innocua; and/or a Staphylococcus spp., preferably S. aureus or S. epididymis.
  • inhibiting the growth and/or proliferation of the at least one bacterial species comprises inhibiting or reducing a bacterial infection, disease and or condition caused by or associated with the bacterial species.
  • the bacterial infection, disease or condition is a bacterial infection, disease or condition of cystic fibrosis, neutropenia, HIV/AIDS, urinary tract infections, community-acquired pneumonia, ventilator-associated pneumonia, endocarditis, meningitis, ocular infections, ear infections, including but not limited to, perichondritis of the ear otitis, externa, malignant otitis externa, and skin and softissue infections, including but not limited to, burns, necrotising fascilitis, ecthyma gangrenosum, green nail syndrome, foot infections, puncture wounds and
  • the combination or composition is formulated as a coating, or is in the form of, a coating or a partial coating.
  • the anti-microbial component is formulated as, or is in a form of, a disinfectant.
  • the object is in inanimate article, material or substance, or part thereof.
  • the object is an object on which bacterial species are known or suspected of being present and/or growing.
  • the object is used in food processing, hygiene, medicine, dentistry or any other industry where contamination by bacterial species poses a health risk and/or is desired to be prevented and/or reduced.
  • the object is selected from the group consisting of medical devices, surgical devices, surgical instruments, surgical implants, stents, catheters, dental devices, dental instruments dental prostheses, dental implants, contact lenses, bandages, wound dressings, and food processing equipment.
  • all of the embodiments set out above that relate to the first or any other aspect of the invention also relate to this method aspect of the invention that is a method of inhibiting the growth and/or proliferation of at least one bacterial species comprising contacting the at least one bacterial species with an anti-microbial component as described herein, wherein the anti-microbial component comprises a polynucleotide in association with an antimicrobial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion.
  • the invention in another aspect relates to a method of treating a microbial infection comprising administering an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, a combination of formula II or Ila, or a composition as described herein to a subject in need thereof.
  • the invention in another aspect relates to a method of treating a microbial infection comprising administering an anti-microbial component to a subject in need thereof, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion.
  • administration is local or systemic administration. In one embodiment, administration is topical, intranasal, epidermal, and transdermal, oral or parenteral. In one embodiment oral
  • administration comprises aerosol delivery to the lungs.
  • parenteral administration is selected from the group consisting of direct application, systemic, subcutaneous, intraperitoneal or intramuscular injection, intravenous drip or infusion, inhalation, insufflation or intrathecal or intraventricular administration.
  • administration is transient administration.
  • transient administration comprises administration of an anti-microbial combination, a targeting component, an anti-microbial
  • Administration can be rapid (e.g., by injection), or can occur over a period of time (e.g., by slow infusion or
  • administration comprises administering a composition or pharmaceutical composition as described herein, wherein the composition or pharmaceutical composition comprises about 100 nM to about 10 ⁇ , or from about 100 nM to 10 ⁇ , or from 100 nM to about 10 ⁇ , or from 100 nM to 10 ⁇ of the anti-microbial combination, targeting component, antimicrobial component, combination of formula I, or combination of formula II or Ila.
  • the concentration is about 1 ⁇ to about 5 ⁇ , or is about 1 ⁇ to 5 ⁇ , or is 1 ⁇ to about 5 ⁇ or is 1 u ⁇ to 5 ⁇ .
  • the concentration is less than 5 ⁇ , about 5 ⁇ or is 5 ⁇ . In one embodiment of the method of treating aspects where the antimicrobial agent is Ag + as described herein, the concentration is less than 1 ⁇ , is about 1 ⁇ , or is 1 ⁇ .
  • administration comprises administering about 100 nM to about 10 ⁇ , or from about 100 nM to 10 ⁇ , or from 100 nM to about 10 ⁇ , or from 100 nM to 10 ⁇ of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila.
  • administration comprises administering about 1 ⁇ to about 5 ⁇ , or is about 1 ⁇ to 5 ⁇ , or is 1 ⁇ to about 5 ⁇ or is 1 u ⁇ to 5 ⁇ .
  • administration comprises administering less than 5 ⁇ , about 5 ⁇ or is 5 ⁇ .
  • administration comprises administering less than 1 ⁇ , is about 1 ⁇ , or is 1 ⁇ .
  • administration comprises administering a sufficient amount of an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, or a
  • the concentration is about 1 ⁇ to about 5 ⁇ , or is about 1 ⁇ to 5 ⁇ , or is 1 ⁇ to about 5 ⁇ or is 1 u ⁇ to 5 ⁇ .
  • the antimicrobial agent is an AgNC as described herein, the concentration is less than 5 ⁇ , about 5 ⁇ or is 5 ⁇ .
  • the antimicrobial agent is Ag + as described herein, the concentration is less than 1 ⁇ , is about 1 ⁇ , or is 1 ⁇ .
  • a sufficient amount comprises about 100 nM to about 10 ⁇ , or from about 100 nM to 10 ⁇ , or from 100 nM to about 10 ⁇ , or from 100 nM to 10 ⁇ of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila.
  • a sufficient amount is about 1 ⁇ to about 5 ⁇ , or is about 1 ⁇ to 5 ⁇ , or is 1 ⁇ to about 5 ⁇ or is 1 u ⁇ to 5 ⁇ .
  • the antimicrobial agent is an AgNC as described herein
  • a sufficient amount is less than 5 ⁇ , about 5 ⁇ or is 5 ⁇ .
  • the antimicrobial agent is Ag + as described herein, a sufficient amount is less than 1 ⁇ , is about 1 ⁇ , or is 1 ⁇ .
  • administration comprises administering a composition or pharmaceutical composition as described herein, wherein the composition or pharmaceutical composition comprises about 1 nM to about 50 ⁇ , or from about InM to 50 ⁇ , or from 1 nM to about 50 ⁇ , or from 1 nM to 50 ⁇ of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila.
  • the concentration is about 1 ⁇ to about 10 ⁇ , or is about 1 ⁇ to 10 ⁇ , or is 1 ⁇ to about 10 ⁇ or is 1 ⁇ to 10 ⁇ .
  • the antimicrobial agent is an AgNC as described herein, the concentration is less than 20 ⁇ , about 20 ⁇ or is 20 ⁇ .
  • the concentration is less than 10 ⁇ , is about 10 ⁇ , or is 10 ⁇ .
  • administration comprises administering about 1 nM to about 50 ⁇ , or from about 1 nM to 50 ⁇ , or from 1 nM to about 50 ⁇ , or from 1 nM to 50 ⁇ of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila.
  • administration comprises administering about 1 ⁇ to about 10 ⁇ , or is about 1 ⁇ to 10 ⁇ , or is 1 ⁇ to about 10 ⁇ or is 1 ⁇ to 10 ⁇ .
  • administration comprises administering less than 20 ⁇ , about 20 ⁇ or is 20 ⁇ . In one embodiment where the antimicrobial agent is Ag + as described herein, administration comprises administering less than 10 ⁇ , is about 10 ⁇ , or is 10 ⁇ . In one embodiment of the method of treating aspects administration comprises administering a sufficient amount of an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, or a
  • the concentration is about 1 ⁇ to about 10 ⁇ , or is about 1 ⁇ to 10 ⁇ , or is 1 ⁇ to about 10 ⁇ or is 1 u ⁇ to 10 ⁇ .
  • the concentration is less than 10 ⁇ , about 10 ⁇ or is 10 ⁇ .
  • the concentration is less than 10 ⁇ , is about 10 ⁇ , or is 10 ⁇ .
  • a sufficient amount comprises about 1 nM to about 50 ⁇ , or from about 1 nM to 50 ⁇ , or from 1 nM to about 50 ⁇ , or from 1 nM to 50 ⁇ of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila .
  • a sufficient amount is about 1 ⁇ to about 10 ⁇ , or is about 1 ⁇ to 10 ⁇ , or is 1 ⁇ to about 10 ⁇ or is 1 u ⁇ to 10 ⁇ .
  • the antimicrobial agent is an AgNC as described herein
  • a sufficient amount is less than 10 ⁇ , about 10 ⁇ or is 10 ⁇ .
  • the antimicrobial agent is an AgNC as described herein
  • antimicrobial agent is Ag + as described herein, a sufficient amount is less than 10 ⁇ , is about 10 ⁇ , or is 10 ⁇ .
  • An anti-microbial combination, a targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila, as described herein may be usefully employed in the methods described when formulated as bioequivalent compounds, including pharmaceutically acceptable salts and prodrugs.
  • Such formulation encompasses any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
  • Pharmaceutically acceptable salts retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • An anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila, as described herein can also be formulated as a prodrug or in prodrug form as known in the art.
  • a prodrug is a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells of a subject by the action of endogenous enzymes or other chemicals and/or conditions.
  • compositions, pharmaceutical compositions and medicaments as described herein, and their subsequent administration following a method of treating a microbial infection as described herein is believed to be within the skill of those in the art.
  • a particular and effective dosage regime will be dependent on severity of the infection to be treated and on the responsiveness of the treated subject to the course of treatment.
  • An effective treatment may last from several hours to several days to several months, or until an acceptable therapeutic outcome is effected or assured or until an acceptable reduction of the infection is observed.
  • An optimal dosing schedule (s) may be calculated from drug accumulation as measured in the body of a treated subject. It is believed to be within the skill of persons in the art to be able to easily determine optimum and/or suitable dosages, dosage formulations and dosage regimes.
  • the optimum dosages may vary depending on the relative potency of a given anti-microbial combination, targeting component, anti-microbial component, combination of formula I, combination of formula II or Ila, or composition as described herein, but will be estimable from an EC50s found to be effective in suitable cells in vitro and in an appropriate in vivo animal model.
  • dosage is from 0.001 g to 99 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, but not limited thereto.
  • the anti-microbial combination, targeting component, anti-microbial component, combination of formula I
  • combination of formula II or Ila, or composition as described herein is administered in conjunction with the administration of an additional anti-microbial agent and/or an additional therapeutic agent.
  • the additional anti-microbial agent is as described herein.
  • the additional therapeutic agent is any appropriate therapeutic agent used to treat or prevent any symptom, side effect or other consequence of treatment, either as a result of the use of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, combination of formula II or Ila, or composition as described herein, in the methods described herein, or for any other reason related to the desired treatment.
  • the additional therapeutic agent is active against microbial infection and/or is used to treat a symptom, side-effect or cause of a microbial infection.
  • anti-microbial combination targeting component, anti-microbial component, combination of formula I, combination of formula II or Ila, or composition as described herein, is formulated with an additional anti-microbial agent and/or an additional therapeutic agent, then the dosing of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I,
  • combination of formula II, or composition as described herein, and the additional therapeutic agent can be separate, simultaneous or concurrent, as is appropriate.
  • Repetition rates for dosing can be based on measured residence times and
  • Maintenance therapy may be desirable in successfully treated patient in order to prevent the recurrence of the infection, wherein the anti-microbial combination, targeting component, anti-microbial component, combination of formula I,
  • combination of formula II or Ila, or composition as described herein is administered in maintenance doses, ranging from 0.001 g to 99 g per kg of body weight, once or more daily, to once every 5 years.
  • the invention in another aspect relates to an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, a combination of formula II or Ila, or a composition as described herein for use in treating, or when used to treat, microbial infection.
  • an anti-microbial component for use in inhibiting the growth and/or proliferation of at least one bacterial species treating, or when used to treat, microbial infection, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an i-motif structure and the anti-microbial agent is at least one metal ion.
  • the invention in another aspect relates to a polynucleotide selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43.
  • the polynucleotide is selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43, and optionally SEQ ID NO: 31.
  • polynucleotide is single stranded. In one embodiment the polynucleotide is comprised in a polynucleotide structure that forms an i-motif. In one embodiment the i-motif comprises at least one of SEQ ID NO: 32, SEQ ID NO: 38,
  • SEQ ID NO: 39 SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43.
  • the i-motif comprises, consists essentially of, or consists of at least one of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43. In one embodiment the i-motif comprises, consists essentially of, or consists of SEQ ID NO: 31.
  • the bacterial strain used for SELEX selection was Pseudomonas aeruginosa 692 (ATCC 14502) obtained from New Zealand Culture Collection (Porirua, NZ).
  • Pseudomonas aeruginosa PAOl ATCC 15692
  • Salmonella enterica serovar typhi ATCC 19430
  • Klebsiella pneumoniae ATCC 13883
  • Enterobacter cloacae ATCC 13047
  • Listeria innocua ATCC 33090
  • Escherichia coli Pseudomonas aeruginosa PAOl
  • Salmonella enterica serovar typhi ATCC 19430
  • Klebsiella pneumoniae ATCC 13883
  • Enterobacter cloacae ATCC 13047
  • Listeria innocua ATCC 33090
  • Escherichia coli Escherichia coli.
  • Additional P. aeruginosa strains used in various assays as described herein were ARL1079, ARL1204, ARL1205, ARL1236, and PA918. These strains are clinical isolates gifted to the inventors by a colleague (D. Day - Personal communication).
  • SELEX Aptamers were selected against live Pseudomonas aeruginosa cells (P. aeruginosa 692 -ATCC 14502) grown as a biofilm using a modified whole-bacterium SELEX [6, 15] protocol.
  • the random ssDNA library (Integrated DNA technologies, USA) consisted of a random 45 nucleotide site flanked by two constant primer regions,
  • ATGAGAGCGTCGGTGTGGTA SEQ ID NO: 33
  • N 4 5-TACTTCCGCACCCTCCTACA SEQ ID NO: 34.
  • the ssDNA library was denatured by heating at 95°C for 5 min then snap cooled on ice for 10 min.
  • 1 nmol at 1 ⁇ of ssDNA was incubated with bacteria in 1 mL binding buffer for 30 min at room temperature (22°C) with gentle agitation.
  • Aptamer bound cells were recovered by centrifugation (3,100xg, 10 min) and washed twice with 1 mL wash buffer to remove unbound oligonucleotides. Washing time and volumes were increased in subsequent rounds to increase the stringency of selection.
  • Bound oligonucleotides were recovered by re-suspending cells in 100 ⁇ L of TE buffer (10 mM Tris-HCI pH 8.0 containing 1 mM EDTA) and heating to 95°C for 5 min then centrifugation at 14,000xg for 15 min. The supernatant that contained the eluted aptamer sequences was kept and used for PCR amplification to generate the next library.
  • TE buffer 10 mM Tris-HCI pH 8.0 containing 1 mM EDTA
  • ATGAGAGCGTCGGTGTGGTA-3' (SEQ ID NO: 35) and 100 nM reverse primer (5'- TGTAGG AGGGTGCGGAAGTA- 3 ') (SEQ ID NO: 36) (IDT, USA) in a total volume of 100 ⁇ L.
  • a thermal cycle consisting of an initial enzyme activation step of 95°C for 10 min, then 95°C for 10 sec and 65°C for 15 sec for the optimised number cycles was used for amplification and the quality of the PCR product was confirmed by agarose gel electrophoresis.
  • the single stranded DNA library was regenerated by asymmetric PCR [16, 17].
  • the asymmetric PCR was performed using 50 ⁇ L of the dsDNA PCR product as template in a final volume of 100 ⁇ L.
  • Amplification was achieved using KAPA2G HotStart DNA polymerase (10 U) (KAPABiosystems, USA) with the supplied KAPA2G buffer A, and contained 0.2 mM dNTPS, 1 ⁇ FAM labelled forward primer (5'-FAM- ATG AG AGC GTC GGTGTG GTA- 3 ') (SEQ ID NO: 37) and 50 nM reverse primer.
  • the PCR was run for 20 cycles using the same cycling conditions.
  • ssDNA PCR product was desalted using an IllustraTM NAPTM-5 column (GE Healthcare, UK) in to wash buffer and stored at -20°C until the next round of selection.
  • ssDNA concentration was determined by 3% agar electrophoresis and densitometry analysis against known concentration standards using ImageJ [18].
  • the evolved aptamer pool was PCR amplified using unmodified primers and purified using the QIAEX II Gel Extraction Kit (Qiagen, Germany). Cloning was performed using the TOPO-TA cloning kit (Thermo Fisher Scientific, USA) following the manufacturer's instructions. White colonies were picked and grown individually overnight at 37°C, before being analysed for the correct sized insert by PCR using the aptamer specific primers. Plasmid from clones containing the desired insert were isolated using the GeneJET plasmid miniprep kit (Thermo Fisher Scientific, USA) following the supplied protocol and were sequenced by Macrogen Inc. (Korea) using the M 13R-pUC primer, and the aptamer sequences were extracted using the Geneious software (Biomatters, NZ). Aptamer candidates were synthesised (IDT, USA) both FAM labelled and unlabelled.
  • Flow cytometry was used to assess binding of the aptamer libraries and the aptamer candidates.
  • FB flow buffer
  • a BD FACSCanto II flow cytometer (BD Biosciences, USA) was used to identify bacteria cells based upon forward and side scatter profiles. Aptamer binding was performed in a volume of 300 ⁇ _ in FB containing 10 ⁇ _ of cells suspension and the desired concentration of library or aptamer.
  • CTC 5-cyano-2,3-ditolyl tetrazolium chloride
  • permeability were determined by staining with propidium iodide (2 ⁇ g/mL, 2 min) and flow cytometry analysis.
  • the dissociation constants were determined by flow cytometry by incubating a set number of bacterial cells with a range of FAM labelled aptamer concentrations from 1 nM to 250 nM . Samples were incubated for 15 minutes at room temperature, collected by centrifugation (13,000xg, 2 min) then re-suspended in 300 ⁇ _ PBS for flow analysis. Binding of aptamers to E. coli cells was used as a measure of non-specific binding and calculation of Kds was done on GraphPad Prism 5 using a one site binding minus non-specific binding model.
  • Aptamers were selected to P. aeruginosa bacteria grown as a biofilm.
  • Whole-cell SELEX was undertaken for seven cycles with three counter-selection steps to exponentially growing P. aeruginosa to bias the selection towards biofilm associated epitopes.
  • Flow cytometric analysis of fluorescently labelled aptamer pools was carried out to assess the binding affinity of each library.
  • the seventh library showed binding enrichment to target cells (Fig. 21) as well as potential metabolic effects and was cloned in to E. coli. 27 different clones were identified by sequencing. Screening of aptamer candidates
  • Single-stranded aptamer candidates were prepared from the plasmids by asymmetric PCR using a fluorescently labelled primer to allow analysis by flow cytometry, and changes in metabolic activity and membrane permeability were determined by staining with CTC and PI respectively.
  • the aptamer candidates were ranked according to binding affinity and their ability to alter metabolic activity or membrane permeability.
  • Two candidates, JN08 and JN27, were chosen to be chemically synthesised and investigated further.
  • the secondary structures of the aptamers were determined using mfold [20] and the secondary structure of JN27 indicated a single stem-loop as being the only significant stable structure, while JN08 contained two putative stem-loops.
  • the stem-loop of JN27 (SEQ ID NO: 1) was hypothesised as being important for binding so a truncated version comprising just the stem-loop (JN27-SH, SEQ ID NO: 3) was synthesised and its binding compared to the full length aptamers.
  • the truncated version showed increased binding when compared to the original length aptamer which confirmed the importance of the stem loop structure (Fig. 2).
  • Kds were determined for all identified aptamers and are displayed in figure 3 and figure 22.
  • JN27-SH displayed a lower Kd than JN27. Without wishing to be bound by theory, the inventors believe that this result supports their hypothesis that the stem- loop motif is important for binding. All Kds are in the low nanomolar range, 10-55 nM .
  • the identified aptamers were evaluated for binding to other bacterial species (Salmonella enterica serovar typhi, Klebsiella pneumoniae, Enterobacter cloacae, Listeria innocua, and Escherichia coli) in comparison with P. aeruginosa PAOl and PA692 by flow cytometry. All aptamers were selective for P. aeruginosa with a very small amount of cross- reactivity with Salmonella and to a lesser extent Enterobacter (Fig. 5, Fig. 23, Table 1).
  • bacteriostatic or bactericidal were determined by changes in PI and CTC staining of the bacterial population, indicative of metabolic changes, after a 15 min incubation with 1 ⁇ aptamer. Preliminary studies showed a change with the enzymatically made aptamers, however the pure, chemically synthesised aptamers did not show any changes in metabolism upon incubation with aptamer. Growth assays were also used to look for an increase in lag phase and time to killing for the aptamer treatments compared to control (Fig. 6). These results were also negative, showing that these aptamers have no intrinsic bacteriostatic and/or bactericidal activity, even though they are highly specific for P. aeruginosa.
  • aptamer binding to Pseudomonas opens doors to potential uses in diagnostics, such as a biosensor for detection of the pathogen in water in hospitals.
  • Pseudomonas forming biofilms inside taps and on equipment is a problem, especially in neonatal wards where an opportunistic infection is usually fatal.
  • These aptamers could be used for the early detection of bacterial contamination.
  • Aptamers are also currently being explored as alternative therapies.
  • the aptamer sequences described herein can be used as targeted therapeutic aptamer conjugates to deliver drugs or other small molecules specifically to the bacteria, increasing the effective concentration at the infection site. This is a problem faced by common antibiotic treatments as the effective concentration is often too low and induces resistance mechanisms. P.
  • aeruginosa is an important bacterial pathogen that needs combatting.
  • the inventors have identified a series of aptamers that bind with high affinity and specificity to P. aeruginosa. These aptamers can be used diagnostically and/or therapeutically by utilising the aptamers as a targeting component as described herein to conjugate to antibiotics or other small molecules for specific delivery to target cells or molecules.
  • Example 2
  • Oligonucleotide DNA templates for nanocluster generation were synthesised by Integrated DNA Technologies (IDT, USA) and were prepared in TE buffer (10 mM Tris- HCI pH 8.0 containing 1 mM EDTA) to a final concentration of 250 ⁇ and stored at - 20°C before use. Sequences used were obtained from our previously reported
  • Nanoclusters were prepared in 10 mM MES buffer (pH 6.5) containing 12.5 ⁇ oligonucleotide template and 75 ⁇ AgNO 3 . The mixture was incubated in the dark at 4°C for 1 hour prior to the slow addition of 75 ⁇ NaBH 4 while the reaction mixture was vigorously mixed by vortexing for 30 seconds. The reaction mixture was then transferred to 4°C and kept in the dark.
  • Fluorescent nanoclusters formed over the next 1-2 hours and were left to mature overnight prior to use. Excess salts and reagents were removed by concentration through an Amicon Ultra-0.5 mL centrifugal filter with a 3000 molecular weight cut-off (Merck Millipore, Germany) to a final volume of approximately 10-15 ⁇ _, followed by dilution with milliQ H2O to 100 ⁇ _. The sample was then concentrated again to a volume of 10-15 ⁇ _ and diluted to a final working volume of 150 ⁇ _.
  • DNA chelated Ag + was generated in a similar manner, they were prepared in 10 mM MES buffer (pH 6.5) containing 12.5 ⁇ oligonucleotide template and 75 ⁇ AgNO 3 . However there was no reduction of the silver ions by NaBH 4 . The silver ions remained bound to the DNA without reduction to silver molecules. Excess salts and reagents were then removed in the same manner with a centrifugal filter as AgNCs, and the final working volume resuspended to 150 ⁇ _.
  • the yield of recovered aptamer was determined by UV-visible spectroscopy.
  • the concentration of aptamer-AgNC or aptamer-Ag + was determined using the extinction coefficient at 260 nm of the native oligonucleotide. All reference to a concentration of aptabiotic used herein refers to this method of concentration determination.
  • the bacterial strains used for aptamer-AgNC and aptamer-Ag + characterisation were P. aeruginosa 692 (ATCC 14502) and PAOl (ATCC 15692) obtained from New Zealand Culture Collection (Porirua, NZ).
  • P. aeruginosa 918 ATCC 27853
  • P. aeruginosa clinical isolates - ARL1024 catheter, urine
  • ARL1205 left thigh
  • ARL1079 tracheal aspirate
  • ARL1236 blood
  • the bacteria were cultured in standard LB medium at 37°C for 16 h with aeration. After growth, the cells were diluted 1 : 100 in fresh LB and grown for 3 hours to allow them to reach exponential phase before use. Bacterial growth assays
  • Growth assays were performed in 96 well microtitre plate format. Growth was monitored by measuring absorbance at 600 nm. Wells containing LB growth media were inoculated with 10 5 CFU of exponentially growing bacteria and treated with aptabiotic or other test substrates. OD 600 readings were taken every 5 minutes for at least 16 hours while the plate was incubated at 37°C. Resulting data was plotted against time to give the bacterial growth curves.
  • Another assay was performed to look at time to killing of the aptamer-AgNC and aptamer-Ag + .
  • a starting inoculum of 2xl0 5 cfu of PA692 in LB was incubated with 1 ⁇ of aptamer-Ag + .
  • Bacteria was incubated with 5 ⁇ aptamer-AgNC or aptamer-Ag + for 30 mins before addition of 2 ⁇ g/mL propidium iodide (PI) to look for changes in cell permeability.
  • the cells were incubated for 2 minutes with PI then changes in cell permeability were determined by flow cytometry analysis.
  • PI propidium iodide
  • Wax moth larvae (Galleria mellonella) were used as in vivo models of infection to test bactericidal and/or bacteriostatic effects of the aptamer-AgNCs and aptamer-Ag + by intrahaemocoelic injection.
  • Fifth instar larvae (130-200 mg in weight) were obtained from Biosuppliers (NZ) and starved for 24 hours before the experiment.
  • a bacterial culture of PAOl was grown as previously described and diluted in lxPBS to a final concentration of 10 3 cfu/mL to get 10 cfu per larvae (in a 10 ⁇ L injection).
  • Larvae were chilled on ice before injection to slow movement and then 10 ⁇ L of prepared bacterial suspension was injected into the right hind proleg.
  • Aptamer-AgNCs or aptamer-Ag + were then administered as a second 10 ⁇ L injection in to the left hind proleg at a concentration of 10, 5 or 2.5 ⁇ .
  • Larvae injected with PBS only were used as a control for injection related mortality. Injected larvae were then incubated at 37°C and checked for death every hour from 12 hours to 24 hours post injection.
  • DNA scaffolded silver nanoclusters have many unique properties, one of these is fluorescence which allows characterisation of the nanoclusters by measuring their excitation and emission spectra.
  • Our Aptamer-AgNC candidates produced emission spectra in the orange to red region of the visible spectrum (Fig. 7). The differences in fluorescent properties could relate to how many atoms of silver are in each cluster and also their association with the surrounding DNA sequence. Fluorescence spectroscopy was also used to measure the stability of the NCs by heating and cooling the same sample to find the temperature at which the NCs became unstable, indicated by a loss of fluorescence, and also whether the NCs would reform when cooled back down, indicated by a return of fluorescence.
  • Circular dichroism spectroscopy was used to determine whether the formation of the silver nanoclusters changes the conformation of the DNA secondary structure.
  • Both JN 17 and JN21 NCs had similar CD spectra to their native oligo before cluster synthesis. This indicates that the formation of the NC on the aptamer sequence does not change the structure significantly and that it appears that the aptamer maintains its secondary structure which is important for binding.
  • Fig. 9 there is a significant change in the CD spectra of JN 17 and JN21 when Ag + is added to the sample in the form of AgNO 3 and left to bind for an hour (Fig. 9).
  • the spectra obtained is characteristic of the formation of an i-motif according to relevant literature [21].
  • I- motifs are characterised as an upwards peak at 290 nm and a downwards peak at 260 nm, also as the concentration of Ag + increases the spectra shifts down and to the right. I-motifs can form in cytosine rich oligonucleotides where strings of C's fold in a certain way.
  • the NC sequences attached to the aptamer sequences are not long enough to form intra-strand i-motifs.
  • the inventors hypothesize the aptamers are coming together as dimers or tetramers to form inter-strand i-motifs with each other in the presence of Ag + .
  • the main aim of this study was to test the antibacterial properties of aptamer-AgNCs and aptamer-Ag + .
  • the aptamers themselves had no effect on the growth of the bacteria so we hypothesised that the targeting ability of the aptamer to P. aeruginosa combined with the silver could make an effective antimicrobial.
  • the aptamer-AgNCs with no linker had a stronger bactericidal effect then those with the linker, irrespective of being on the 5' or 3' side (Fig. 25). Without wishing to be bound by theory, the inventors believe that this result is consistent with the CD spectral analysis shown in figure 9, and supports the hypothesis that the nanoclusters do not significantly alter the aptamer secondary structure.
  • the JN21 aptamer was also more effective as the split NC sequence on both the 5' and 3' side, and this, along with JN 17 split NC, JN27 5'-NC and St21Lpl7 split- NC were our top candidates for further investigation.
  • the aptamer-Ag + compositions were also able to kill a range of different strains of P. aeruginosa other than the test strains PA692 and PA01.
  • a range of strains, as indicated in Fig. 17, were treated with 2.5 ⁇ aptamer-Ag + and the growth monitored by OD600 measurement as previously described.
  • All P. aeruginosa strains were highly susceptible to aptamer-Ag + treatment, compared with the equivalent concentration of AgNOs (Fig. 17, Fig 32).
  • the efficacy of the aptamer-AgNC and aptamer-Ag + was then tested in vivo using Galleria mellonella larvae which is an established invertebrate (insect) model for infection.
  • aeruginosa strain PAOl was used as the infecting bacteria as it is highly virulent being capable of killing larvae at low doses.
  • Larvae were injected with the bacteria followed by a second injection containing the treatment after 15 minutes.
  • a comparison between larvae treated with lower dose of 5 ⁇ JN21 aptamer-AgNC or JN21 aptamer-Ag + showed an increase of 4.5 hours to the median survival time for those treated with the aptamer-Ag + (Fig. 18).
  • Flow cytometry was performed using a BD FACSCanto II flow cytometer (BD
  • a 10 ⁇ L aliquot of cells (5xl0 4 CFU) was incubated with 5 and 10 ⁇ aptamer-AgNC, or a range of aptamer-Ag + concentrations (5 ⁇ to 0.078 ⁇ ) each in a total volume of 50 ⁇ L for 10 min or 1 hr before addition of 1.5 ⁇ g/mL propidium iodide (PI) and 1.5 mM SYT09.
  • PI propidium iodide
  • SYT09 1.5 mM SYT09.
  • Cells were identified by their forward and side scatter properties and by staining with 1.5 mM SYT09. At least 10,000 events in the gated bacterial population were collected and the data analysed using Flowing Software 2.5.1 (Turku Centre for Biotechnology, Finland).
  • I-motifs can form in cytosine rich oligonucleotides where strings of cytosines fold in a certain way.
  • the NC sequences attached to the aptamer sequences are not long enough to form intra-strand i-motifs.
  • the inventors hypothesize the aptamers are coming together as dimers or tetramers to form inter-strand i-motifs with each other in the presence of Ag + .
  • Aptamers JN21 split, JN27 5' and st21lpl7 split identified as forming i-motifs in the presence of Ag + were tested for antibacterial activity, as the Ag + remained stably associated with the DNA after removing the excess silver from the sample (Fig. 13 and Fig. 24).
  • the term "aptamer-Ag + " as used herein means aptamers that form i-motifs that remain stably associated with Ag + .
  • aeruginosa without treatment (black solid), when treated with 2.5 ⁇ AgNC (dark grey dashed), or when treated with 2.5 ⁇ of the same nanocluster forming sequence conjugated to either aptamer JN27 (dark grey solid), JN21 (light grey solid) or St21Lpl7 (black dashed).
  • Treatment with the untargeted AgNC results in a significant increase in the time required for the culture to achieve the same density as the untreated control (arrowed line). The growth delay is greater for all of the aptamer conjugated AgNCs relative to the untargeted AgNC indicating enhanced killing due to aptamer targeting.
  • Figure 26 shows the effect that a 2-fold dilution series in the range 10 ⁇ to 78 nM of different aptamer-AgNCs has on the growth of PA692 (three independent experiments were performed and similar results were obtained for each). Concentrations were based on the concentration of the DNA, assuming that each synthesis is the same when following the same standard protocol. In the absence of treatment (black solid) cultures grew rapidly and achieved an optical density greater than 0.1 by
  • aptamer-Ag + had strong antibacterial activity (Fig. 24). All aptamer-Ag + tested showed no growth at 2.5 ⁇ after 20 hours of culture. Enumeration of viable CFU was performed for the 2.5 ⁇ treatment after 20 hours to determine the extent of killing. JN27 5' showed 99.996% killing, St21lpl7 3' 99.995% killing and JN21 split 99.7% killing (Fig. 14). To determine whether no growth in the clear wells at higher concentrations was due to the length of the experiment or complete sterilisation, the whole wells were spread on to agar plates.
  • an inoculum containing 2xl0 5 CFU was treated with 5 or 10 ⁇ of JN27 5', JN21 split or St21Lpl7 split-AgNC, or 1 ⁇ and 2.5 ⁇ of the same aptamers made as aptamer-Ag + rather than reduced to AgNCs. After 10 min, 1 hr and 3 hrs, traces of aptamer-AgNC/Ag + were removed by dilution then plated for enumeration of CFU.
  • aptamer-Ag + 1 ⁇ treatment resulted in approximately 50% bacterial death for JN27 and JN21 (49.4 ⁇ 8.7% and 50.7 ⁇ 10.2%) after 10 min, with 23.2 ⁇ 7.3% for St21Lpl7. After 1 hr these aptamer-Ag + s had killed 83.1 ⁇ 4.7%, 80.1 ⁇ 4.4%, and 89.5 ⁇ 7.7% respectively. At 3 hrs they had killed 98.4 ⁇ 0.3%, 98.1 ⁇ 0.5%, and 99.8 ⁇ 0.1% of the starting population respectively (Figure 34, table 2).
  • aptamer-AgNC and aptamer-Ag + treatment were explored using flow cytometry to look at the permeabilisation of the cells using the viability stain propidium iodide.
  • One mode of silver toxicity is through disruption of membrane integrity and disruption of electron transport leading to a decreased proton motive force (pmf) for ATP synthesis and other energy requiring processes.
  • the vital dye PI is excluded from healthy bacterial cells because of its low membrane permeability and extrusion by pmf energised efflux pumps; dead, damaged or de-energised bacteria show an increase in PI uptake that can be readily measured by flow cytometry.
  • Exponentially grown P. aeruginosa when washed and stained with PI can be fractionated into two populations, those that are highly impermeable and show high levels of PI extrusion, and those that have increased permeability.
  • Figure 38A and 38B shows larvae injected with PAOl and then treated with three different aptamer-AgNCs at 10 ⁇ to look for an increase in median survival time compared to untreated control.
  • Two independent experiments were performed and similar results obtained for each. Each treatment was given as a second injection 20 min following infection with 10 CFU of PA01. All larvae for the untreated control were dead by 20 hours (black solid).
  • Treatment with JN21 split-AgNC (black dashed) and st211 p 17 split-AgNC (grey dashed) increased the median survival time to 19 hours, compared to 18 hours for control, and JN27 5'-AgNC (grey solid) increased median survival to 20 hours.
  • Aptamer-AgNC and aptamer-Ag + conjugates are not substrates for efflux pumps
  • P. aeruginosa has high resistance to many antibiotics through expression of multidrug resistance pumps of the RND class [31, 32, 33]. These broad specificity pumps are able to extrude antimicrobials such that the intracellular concentration does not achieve a sufficient therapeutic dose.
  • Panel A shows a dose response plot for PA01 (black) and MexB (red) treatment with ciprofloxacin for 16 hrs
  • panel B-D treatment with each aptamer-AgNC for 16 hrs.
  • the efflux mutant is highly sensitive to ciprofloxacin with the IC50 being 0.01781 ⁇ g/mL, compared to 0.2507 ⁇ g/mL for wild type PA01, whereas the aptamer-AgNCs and aptamer-Ag + s had similar sensitivity in the MexB mutant and wild type strain.
  • Aptamer-AgNC and aptamer-Ag + conjugates cause dissociation of established biofilms
  • Aptamer-AgNCs and aptamer-Ag + s were tested as a treatment (5 ⁇ ) against both immature (20 hour) and mature (44 hour) biofilms and their action compared to that of the common fluoroquinolone antibiotic ciprofloxacin at lxMIC or 0.5xMIC for planktonic PA692. Immature biofilms were treated either once for 6 hours, or twice with the second treatment lasting 17 hours. This was to see if the replacement of the aptabiotic with fresh aptabiotic to keep the concentration high would increase the killing effect as it is hypothesised that the aptabiotic degrades during treatment.
  • Circular dichroism spectra were performed as in example 3, except a 6-fold molar excess of Ag + was added rather than a titration performed. A 12-fold molar excess was required in once instance, as indicated, to form the i-motif.
  • FIG 43 shows that the Ag + stabilised i-motif (SEQ ID NO 38) has bactericidal or bacteriostatic activity against both Gram-negative bacteria and Gram-positive bacteria. Complete inhibition of growth was achieved at a concentration of 2.5 ⁇ for P. aeruginosa, E. coli, and K. pneumoniae, (Fig 43A, B, D, F) while the same concentration suppressed growth of S. aureus, L. innocua, E. cloacae, S . epidermidis, S. enterica (Fig 43C, E, G H I). Growth inhibition was observed at all concentrations of the Ag + stabilised i-motif tested.
  • Table 4 records the time required for the culture to attain an OD600 of 0.1 as a measure of the extent of growth inhibition relative to the untreated control. Similar results would be expected if other Gram-positive and Gram- negative were tested for susceptibility. Table 4. The time taken (minutes) for different bacterial species treated with a range of concentrations ( ⁇ ) of i-motif2 (SEQ ID NO:38) to reach a threshold value of 0.1 OD600- Dashes indicate the threshold was not met during the 1015 minute duration of the experiment.
  • Sequences were selected from a set comprising poly-C tracts and turns, such that the i-motif is formed by concatermerisation of alternating poly-C and turns.
  • the poly-C tracts were selected from a set that contained the sequences: CCC, CCCC, CCCCC, CCCCCC, CCCCCCC, CCCCCCCC, CCCCCCCCC, and the turns from the set: A, T,
  • Figure 44 and Figure 45 show that all the Ag + i-motif sequences tested inhibited the growth of P. aeruginosa strains PA692 and PAOl respectively, but that the i-motif forming sequences differ in their antimicrobial activity.
  • Table 5 shows that all seven of the different Ag + stabilised i-motifs tested were either bacteriostatic or bactericidal for P. aeruginosa strain PAOl at the concentrations tested .
  • Table 5 records the time required for the cultures to obtain an OD 600 of 0.1. No growth was observed after 1015 minutes in culture for any of the Ag + i-motifs at 5 ⁇ concentration. Table 5.
  • the time taken (minutes) for P. aeruginosa strain PAOl treated with a range of concentrations of different i-motif structures to reach a threshold value of O.l OD600- Dashes indicate the threshold value was not met during the time 1015 period and no growth occurred.
  • Table 6 shows that all seven of the different Ag + stabilised i-motifs tested were either bacteriostatic or bactericidal for P. aeruginosa strain PA692 at the concentrations tested .
  • Table 6 records the time required for the cultures to obtain an OD600 of 0.1. No growth was observed after 1015 minutes in culture for any of the Ag + i-motifs at 2.5 and 5 ⁇ concentrations.
  • Figure 47 shows that all the Ag + i-motif sequences tested had an inhibitory effect on the growth of Staphylococcus aureus, and that the i-motif forming sequences differ in their antimicrobial activity.
  • Table 7 shows that all seven of the different Ag + stabilised i-motifs tested were either bacteriostatic or bactericidal for S. aureus at the concentrations tested. Table 7 records the time required for the cultures to obtain an OD600 of 0.1. Table 7. Time taken (minutes) for S. aureus treated with a range of
  • Table 8 Time taken (minutes) for different bacterial species treated with 1.25 ⁇ of i-motif2 (SEQ ID NO:38), with or without 2 mM EDTA co- treatment, to reach a threshold value of 0.1 OD600 . Dashes indicate the threshold value was not met during the time period (1015 minutes) of the experiment and no growth occurred . Bacteria were treated with 2 mM EDTA alone also as a control.
  • S .aureus also showed strong anti-microbial synergy between EDTA treatment and half i-motifl (SEQ ID NO: 39).
  • the half i-motifl was used in the concentration range 0.078 ⁇ to 1.25 ⁇ , with EDTA in the range 0.1 mM to 0.75 mM.
  • Figure 49 shows growth curves for the various combinations of half i- motifl and EDTA, and demonstrates that EDTA enhances the antibacterial effect of half i-motifl above that of EDTA treatment alone, or half i-motif treatment alone. Table 9. Time taken (minutes) for S.
  • JN08 split NC - 5'-TCC CCC ATG CAC TCT ATG TAG GAG GGT GCG GAC CCC T-3' SEQ ID NO: 11 JN21.SH - 5'-AGA GCG TCG GTG TGG TAA CTG TTC AGG AGG ATG ACA TTG TCG CCT-
  • St21Lpl7 split NC- 5'- TCC CCC AAG CGT CGG TGT TCT ATC GGT AGT TGA CAC CGA CGC CTC CCC T-3'
  • Stl7Lp21 split NC - 5'-TCC CCC ATC CTC CTA GGT AAC TGT TCA GGA TGT AGG AGG GTC CCC T-3' SEQ ID NO: 27
  • St08Lpl7 split NC - 5'-TCC CCC ATG CAC TCT TCT ATC GGT AGT TGA GGG TGC GGT CCC CT-3'
  • Reverse primer flanking region - 5'-TAC TTC CGC ACC CTC CTA CA-3' SEQ ID NO: 35
  • Forward primer for PCR - 5'- ATG AGA GCG TCG GTG TGG TA-3' SEQ ID NO: 36
  • anti-microbial combinations, targeting components, anti-microbial components and anti-microbial combinations disclosed herein all have industrial application as bacteriostatic and/or bactericidal agents, can be formulated into conjugates, complexes and compositions having bacteriostatic and/or bactericidal activity, and can be used in both the manufacture of medicaments for, and the direct treatment of, microbial infection.
  • Pseudomonas aeruginosa biofilms Towards the development of novel anti- biofilm therapies," J. Biotechnol., vol. 191, pp. 121-130, 2014.
  • RNA ligands to bacteriophage T4 DNA polymerase Science (80-. ). , vol. 249, no. 4968, pp. 505-510, 1990.

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Abstract

La présente invention concerne des agents antimicrobiens (AMA) présentant des propriétés bactériostatiques et/ou bactéricides, des compositions et des combinaisons comprenant un AMA et l'utilisation de telles compositions, combinaisons et/ou de tels AMA pour inhiber la croissance et/ou la prolifération de micro-organismes, à utiliser dans la fabrication de médicaments destinés au traitement d'une infection microbienne et/ou à utiliser dans le traitement d'une infection microbienne.
PCT/IB2018/052437 2017-04-07 2018-04-09 Combinaison antimicrobienne WO2018185735A1 (fr)

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WO2024225895A1 (fr) * 2023-04-24 2024-10-31 Universiteit Leiden Agent cytotoxique

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2822623C1 (ru) * 2023-03-20 2024-07-10 Федеральное государственное бюджетное учреждение науки Сибирский федеральный научный центр агробиотехнологий Российской академии наук (СФНЦА РАН) Способ исследования снижения биопленкообразования Escherichia coli лечебной композицией, содержащей наночастицы серебра и ципрофлоксацин
WO2024225895A1 (fr) * 2023-04-24 2024-10-31 Universiteit Leiden Agent cytotoxique
NL2034670B1 (en) * 2023-04-24 2024-10-31 Univ Leiden Cytotoxic agent

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