HHMI at AVI BioPharma
PMOs as Antibiotics: Genespecific Inhibition of E. coli
Mentor: Dr. Bruce Geller
Susan Puckett
Background
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
Antibiotics today— a
biological arms race
Some bacteria have
developed resistance
and immunity to
available antibiotics
 MRSA, TB,
Pseudomonas,
VRE
Overview
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AVI BioPharma, OSU-LARC
Using man-made DNA mimics called
phosphorodiamidate morpholino oligomers
(PMOs) to block production of essential
protein in E. coli
PMOs work as antibiotics to inhibit bacterial
growth
Antisense mechanism: forming
complementary base pairs with nucleic acid
to change gene expression
Escherichia coli

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bacterium, prokaryote
E. coli is a model organism
 A lot of information
available
 Easy to obtain, work with
and grow
 Safe (few dangerous
strains, many harmless
unless in large quantities)
We are trying to find ways to
inhibit the growth of E. coli
using DNA mimics (PMOs)
Eventually this technology
can be used with other
disease causing organisms
Phosphorodiamidate
Morpholino Oligomers (PMOs)


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DNA mimics
Structure is different
enough so that
nucleases don’t break
it down
Used to anneal to
mRNA in order to
block ribosomes from
attaching, disrupting
protein synthesis
PMOs can be
manufactured to
match RNA to form
complementary base
pairs in the same
manner nucleic acids
bind to each other
PMO: a picture
RNA strand: 5’……….AUG AGC ACU AUC GAA GAA CGC GUU………..3’
PMO:
G TGA TAG CTT C
1)
A
U
G
A
G
RNA
PMO
C
A
C
U
A
U
C
G
A
A
G
G
T
G
A
T
A
G
C
T
T
C
C
A
C
U
A
U
C
G
A
A
G
G
T
G
A
T
A
G
C
T
T
C
A
2)
A
U
G
A
G
A
Protein Synthesis
Three Stages
Initiation of Translation
Elongation
Termination
In the initiation of translation stage, the ribosome finds the ribosome binding
site (RBS) on the mRNA and attaches.
PMOs are thought to prevent the two parts of the ribosome from coming
together and attaching to the mRNA.
PMOs and RNA

What size of PMO works best? What
gene in E. coli to target? What locus on
the gene to target?
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11 base pair PMO most effective for
inhibiting translation
AcpP gene necessary for production of
essential protein
Targeting just downstream of the ribosome
binding site
PMOs and Peptides
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Problem: getting PMO
into cell
Peptides attached to
PMOs increase the
amount of compound
that enters the cell
Question: gene specific
or general toxicity?
Experiment: showing
specificity
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Wildtype (original) strain of E. coli (W3110)
and mutant strain of E. coli in the AcpP gene
(LT 1.7) mixed with AcpP PMO, mutant PMO,
scrambled PMO, or water.
Incubated, recorded optical density (OD600)
using microplate reader every hour to
measure growth.
Optical Densities vs. Time
(Growth Curves)
5’….AUG AGC ACU AUC GAA GAA CGC GUU…..3’
G TGA TAG CTT C
AcpP mRNA
AcpP PMO
5’….AUG AGU ACC AUU GAG GAA CGC GU…..3’
A TGG TAA CTC C
AcpP mutant mRNA
AcpP PMOmut4
Conclusion of experiment
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Order of bases matters: sequence specific
inhibition
PMOs do inhibit bacterial growth
Experiment: Minimum
Inhibitory Concentration (MIC)
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How much PMO is needed to prevent growth of E. coli?
Experiment tests the ability of different concentrations of peptidePMOs to inhibit strains of E. coli.
PMO and bacteria are combined in a 96-well plate, incubated
overnight, and examined.
Results
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Various PMOs tested as well as some traditional
antibiotics such as tetracycline, rifampin
Example: MIC determined to be 2.5 uM (micromolar)
for the (RFF)3R-AcpP PMO
optical
density
readings

40 uM
20 uM
10 uM
5 uM
2.5uM
1.25 uM
0.004
0.000
0.001
-0.001
0.575
0.451
0.003
0.001
0.001
0.571
0.000
0.119
0.003
0.015
0.000
0.000
0.000
0.410
Watch for contamination, mutants
Isolation of Mutants
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Growth at the MIC for (RFF)3R-AcpP PMO was used
for a second MIC
Significantly higher MIC
Streak plated
Colonies were picked and named W3110R1 through
10 (W3110 was the parent strain)
Gram stain tests
Sequencing of AcpP target region
Frequency of mutation: 1.4 x 10-6 on plates with
20uM PMO
Evidence of peptide related
mutation
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No change in sequence of AcpP target
Targeting a different gene with the same
peptide still has resistance
Therefore we think the mutation must be
affecting the peptide uptake into the cell
This is useful because very little is known about how
the peptide gets into the cell.
FtsZ Graphs
0 uM FtsZ PMO 08-01-06
FtsZ: another gene in E. coli essential
for protein production
1.00E+11
1.00E+10
1.00E+09
CFU/mL
1.00E+08
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
160 uM FtsZ PMO 08-01-06
1.00E+00
W3110R1
W3110 parent
E. coli
1.00E+11
1.00E+10
1.00E+09
1.00E+08
CFU/mL
Colony-forming unit (CFU) per mL:
found from plate counting
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
W3110R1
W3110 parent
E. coli
Minimum Inhibitory Concentrations
45
40
Concentration (µM)
35
30
25
W3110
W3110R
20
15
10
5
0
(RFF)3RAcpP11
D-(RFF)3RAcpP11
(KFF)3KAcpP11
(RXX)3-AcpP
PMO
(RTR)-AcpP11
(RXR)4AcpP11
(RX)6BAcpP11
Conclusion
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(RFF)3R-AcpP sequence specific inhibition
MIC of (RFF)3R-AcpP is 2.5 uM
Mutants can be isolated and mutants appear
to be peptide related
Future experimentation
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Identifying mutant gene in
resistant strain
Other bacteria
Testing in vitro and in vivo
Acknowledgements
Dr. Bruce Geller
Luke Tilley
Brett Mellbye
AVI BioPharma
Dr. Kevin Ahern
Howard Hughes Medical Institute
Publications: Lucas D. Tilley, Brett L. Mellbye, Susan E. Puckett, Patrick.
L. Iversen, Bruce L. Geller. 2006. Antisense Peptide-Phosphorodiamidate
Morpholino Oligomer Conjugate: Dose-Response in Mice Infected with
Escherichia coli. J. Antimicrob. Chemother. In press.