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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

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% 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

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80

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100

% E

mbr

yos

Surv

ivin

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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.