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Photoactivated RNAi with Caged 2'Fluoro-RNA in Cells and Tissues
Rick Blidner1, Kurt Svoboda2, Bob Hammer 3,Todd Monroe1
1Department of Biological and Agricultural Engineering2Department of Biological Sciences
3Department of ChemistryLouisiana State University, Baton Rouge, LA
IBE ConferenceChapel Hill, NCMarch 6-9, 2008
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Project Overview• Photo-control of RNA interference • FNAs : stable RNA analogs• Adds controllable trigger to gene knockdown• Also adds resistance to enzymatic digestion• Demonstrated in:
– Cell culture GFP assay
– Zebrafish embryo GFP and development assay
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Caged Molecules
• Photo-activation of biological activity• Study time course of cellular responses
N
N
NNH2
N
O
OHHO
OPO
O-POO
O-POO
O-O
N
N
NNH2
N
O
OHHO
OPO
O-POO
O-P-OO
O-O
Released Cage(DMNAP)
O
NONO2
Released ATPγ-phosphate caged ATP
hn +
Kaplan, J. H., B. Forbush, et al. (1978). Biochemistry 17(10): 1929-1935.
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RNA InterferencesiRNA introduction
RLC loading (optional)
RISC assembly
RISC maturation
target recognition
target cleavageandrelease
mature RISCrecycling
siRNA introduction
RLC loading (optional)
RISC assembly
RISC maturation
target recognition
target cleavageandrelease
mature RISCrecycling
John Casey
• Post-transcriptional gene silencing from small regulatory RNAs
• More effective than antisense agents
• Endogenous miRNAs are critical in gene regulation and development
• 2006 Medicine Nobel to A. Fire and C. Mello for discovery of RNAi
• Needs control for mechanistic studies, targeting for therapeutics
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Therapeutic Application
365nm laser light
2. Non-specifically Deliver Caged (Inactive) therapeutics
Localized Sites of Disease/Dysfunction
3. Activate Caged RNA with Pinpointed Light Exposure
4. Active si/shRNA have therapeutic affects in desired locations
Cleavagesite
1. Chemically Inactivate RNAi precursor
OR
PO
O
O-
O
OHO
HH
HH
PO
OR
O-
O
OHO
HH
HH
PO
O
O
NO 2
H3CO
H3COBase
Base
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Initial Studies: Caging Protocol
Parameter Study• Cage concentration• Temp, time of rxn• Solvent, pH conditions
Evaluation Study• Cage attachment• Oligo purification
NO2
N N
BASEO
OHO
'5
P-O O
BASEO
OHO
O
PO O
BASEO
OH3'
ONO2
BASEO
OHO
'5
P-O O
BASEO
OHO
O
P-O O
BASEO
OH3'
O
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Initial Studies: 700bp dsRNA Caging
Caged
Control + Light
Caged
Control + LightControl
Caged
Control + Light
Caged
Control + LightControl
Caged + Light
Cage + Light
Caged + Light
Cage + LightControl + Light
Caged + Light
Cage + Light
Caged + Light
Cage + LightControl + Light
BASEO
O
OP O-OO
BASEO
OH
5'
OP O-OO
BASEO
OH
OP OOO
R
3'
H
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Confirmation of Caged 2’FNA
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Photo-induced RNAi Experiments
caged nucleicacid + plasmid
CationicLiposomes Lipoplexes
+
Measure GFP Expression
Cultured BHK Cells
365nm light
(dark) (photoactivated)
Caged siFNA Uncaged siFNA
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RNAi with caged siFNAs
• Mix and Match– Control Antisense– Control Sense– Caged Antisense– Caged Sense
• Caged– ↓ RNAi activity
• Photoexposed– ↑ RNAi activity
Perc
ent C
ells
with
RN
Ai R
espo
nse
(%)
0
20
40
60
80
100 siFNA no light treatmentsiFNA + 365 nm light
siFNAA.ControlS.Control
siFNAA.CagedS.Control
siFNAA.ControlS.Caged
siFNAA.CagedS.Caged
†
†
†
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Zebrafish Injections• Co-injection of all reagents
– Plasmid– siFNA (control or caged)– Tracking dye (conjugated dextran)– Phenol red
• 28 hpf evaluation – Fixed and evaluated by microscopy– Tracking dye used to select good injections– Each experiment had GFP control fish
Alexa Fluor 55410,000 MW Dextran
AcGFP-N1 MergedAlexa Fluor 55410,000 MW Dextran
AcGFP-N1 Merged
Zfin book
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GFP Knockdown:Native and Caged siFNAs
GFP-control siFNA + GFP siFNA + GFP + UVA Light
Caged siFNA + GFP + UVA Light
Caged siFNA + GFPGFP-control
A
B
GFP-control siFNA + GFP siFNA + GFP + UVA Light
Caged siFNA + GFP + UVA Light
Caged siFNA + GFPGFP-control
A
B
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Toxicity: Native and Caged siFNAs
• siFNA titration shows toxicity
• Caging reduces the observed toxicity
• Characterized by delayed development
control
native 500 ng si-FNA
caged 500 ng si-FNA
10/40
43/67
7/41
0
10
20
30
40
50
60
70
80
90
100
% E
mbr
yos
Surv
ivin
g to
24
hpf
500ng/µL pAcGFP500ng/µL siFNA
250ng/µL pAcGFP500ng/µL siFNA
125ng/µL pAcGFP250ng/µL siFNA
0 3 6 9 12 15 18 21 24 27 300
20
40
60
80
100
% embryos survived
age (hpf)
control injection
caged siFNA
native siFNA
% E
mbr
yos
Surv
ivin
g
control
native 500 ng si-FNA
caged 500 ng si-FNA
10/40
43/67
7/41
0
10
20
30
40
50
60
70
80
90
100
% E
mbr
yos
Surv
ivin
g to
24
hpf
500ng/µL pAcGFP500ng/µL siFNA
250ng/µL pAcGFP500ng/µL siFNA
125ng/µL pAcGFP250ng/µL siFNA
0 3 6 9 12 15 18 21 24 27 300
20
40
60
80
100
% embryos survived
age (hpf)
control injection
caged siFNA
native siFNA
% E
mbr
yos
Surv
ivin
g
500ng/µL
500ng/µL
caged 500 ng siFNA
Native 500 ng siFNA
control
native 500 ng si-FNA
caged 500 ng si-FNA
10/40
43/67
7/41
0
10
20
30
40
50
60
70
80
90
100
% E
mbr
yos
Surv
ivin
g to
24
hpf
500ng/µL pAcGFP500ng/µL siFNA
250ng/µL pAcGFP500ng/µL siFNA
125ng/µL pAcGFP250ng/µL siFNA
0 3 6 9 12 15 18 21 24 27 300
20
40
60
80
100
% embryos survived
age (hpf)
control injection
caged siFNA
native siFNA
% E
mbr
yos
Surv
ivin
g
control
native 500 ng si-FNA
caged 500 ng si-FNA
10/40
43/67
7/41
0
10
20
30
40
50
60
70
80
90
100
% E
mbr
yos
Surv
ivin
g to
24
hpf
500ng/µL pAcGFP500ng/µL siFNA
250ng/µL pAcGFP500ng/µL siFNA
125ng/µL pAcGFP250ng/µL siFNA
0 3 6 9 12 15 18 21 24 27 300
20
40
60
80
100
% embryos survived
age (hpf)
control injection
caged siFNA
native siFNA
% E
mbr
yos
Surv
ivin
g
500ng/µL
500ng/µL
caged 500 ng siFNA
Native 500 ng siFNA
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Summary of Caged siFNA Results
• Cage protects from nuclease digestion• Cell culture
– Cage blocks RNAi activity– Caging antisense strand is more effective– Photo-exposure partially restores activity
• Fish embryos– Cage blocks RNAi activity– Photo-exposure partially restores activity– Cage can protect biological system from toxicity of
high effector concentration
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Acknowledgements • Collaborators:
– Hammer, Svoboda Labs at LSU– Julianne Audiffred, Chris Kennedy, John Casey, Leah
Muller, Megan Harris
• Support:– National Science Foundation
• Grant Number EPS-0346411 (CBM2)• NSF CAREER Award Program
– LSU Ag. Center Biotech Program– Invitrogen, Inc.