SM
NOVEL TECHNOLOGY:
asymmetric siRNA Duplexes
[A-siRNA]
Novel siRNA with chemical and structural modifications
to ensure asymmetric and potent RNAi activity.
ADVANTAGES OVER TYPICAL siRNA:
Greater Specificity of Suppression
Reduce Off-Target Suppression
Reliable for Drug Development
Degradation Resistant invitro/invivo
Superior Design and Chemistry
www.oligoengine.com
Asymmetric siRNA Duplex:
Right on target for Research and Therapeutics.
The A-siRNA Duplex was developed in 2003 by Oligoengine scientists as a more
precise RNAi trigger than dsRNA based siRNA. The structure contributes to the
asymmetric loading into RISC and the inactive sense strand prevents ‘sense-strand’
based off-target effects caused by canonical siRNA.
A-siRNA Duplexes have been used to specifically inhibit the expression of proteins
in vitro, such as the GL2 luciferase protein. The A-siRNA Duplex is especially useful
for in vivo studies and therapeutics development (see inset on opposite page).
5’ Mod
inactive template
Blunt
clamp
active guide RNA
dTdT
Suppression of Firefly Luciferase Exppression
100%
100
Percent of GL2 Expression
(relative to control)
Effective Knockdown
To test the effectiveness of the A-siRNA
Duplex in targeted gene suppression, an
A-siRNA Duplex was designed against
GL2 form of the firefly luciferase and tested
in 3T3-Lux cells. As shown at right, GL2
expression was suppressed over 90% with
the A-siRNA Duplex compared to a control
lacking siRNA. Multiple runs of this same
experiment demonstrated that RNAi
directed by A-siRNA Duplexes are
effectively repeatable and titratable.
1 = Control (cells only)
2 = 25 nM A-siRNA Duplex
3 = 50 nM A-siRNA Duplex
4 = 100 nM A-siRNA Duplex
5 = 200 nM A-siRNA Duplex
75
50
41%
39%
25
20%
5%
0
1
2
3
4
5
Based on fast, proven, and scalable synthesis techniques, the A-siRNA technology
provides an ideal platform for commercial applications. The dependability and costeffectiveness of A-siRNA synthesis for target discovery and genome-wide RNAi
applications supports the use of “just-in-time” arrays to vastly improve the drug
development process.
Transfection of luciferase-expressing 293 cells with siRNA or A-siRNA
against the luciferase gene
Invitro Comparison
Luciferase Activity
200000
200000
180000
180000
160000
160000
140000
140000
120000
120000
GL2 A-siRNA
100000
100000
GL2 siRNA
80000
80000
GFP siRNA
60000
60000
40000
40000
20000
20000
00
1 fmol
1fmol
4 4fmol
fmol
2020fmol
fmol
100
fmol 500
fmol 20
pmol
100fmol
500fmol
20pmol
Oligoengine
CORPORATE OFFICE
1409 42nd Ave. E.
Seattle, WA 98112
A-siRNA
siRNA
LABORATORY
Seattle, WA
Phone:
Fax:
Toll-free:
(206) 254-0200
(206) 254.0300
(800) 516.5446
A-siRNA
Asymmetric siRNA Duplex
TM
Removing off-target effects from RNAi
To prove the negative effect of typical siRNA, Oligoengine first designed an siRNA
that shared GL2/GL3 complimentarity in both the sense and antisense strands. The
ability of an siRNA to function in either direction would result in an undesirable
suppression of GL3.
The same construct was tested using the Asymmetric siRNA composition which
illustrated the benefit of de-activating the sense strand (template) of siRNA and
prevention of sense-strand based off-target suppression.
Comparison of Specificity: A-siRNA Duplex vs. siRNA
Relative GL3 Luciferase Expression
Eliminates off-target Suppression
To demonstrate the specificity of the targeted
A-siRNA Duplex response, NIH3T3 fibroblast
cells that stably express the GL3 form of the
firefly luciferase were transfected with varying
concentrations of A-siRNA Duplex and
chemically synthesized siRNA – both targeting
the same region of the GL2 luciferase mRNA.
In contrast to the A-siRNA Duplex, which
showed no significant knockdown of GL3
luciferase expression, the siRNA demonstrated
an “off-target” suppression of the GL3 protein,
as shown in the chart at right.
120
100
•
•
•
80
•
•
•
•
•
A-siRNA: GL2
• • • • • •
•
• •
•
• •
siRNA: GL2
60
40
20
0
0
5
10
15
20
25
A-siRNA/siRNA Concentration (nM)
Additional genome-wide assays have shown the specific signature of A-siRNA.
Efficient Processing: A-siRNA Duplex vs. siRNA
A-siRNA vs. siRNA processing
Asymmetric Duplex
A-siRNA provides advantages
siRNA
of potency and specificity when
compared to other RNAi
3’ -Sense DNA
5’ -Antisense RNA
Modified 5’
3’
methods.
Active RISC
5’
3’
3’ -Sense RNA
5’ -Antisense RNA
5’
3’
reverse in PAZ
Active RISC
3’
5’
5’
3’
3’
5’
3’
5’
5’
3’
vs.
Target
Recognition
Target
Recognition
5’
5’
Primary mRNA Target
3’
3’
Primary mRNA Target
3’
Off-Target
Recognition
Cleaved Primary mRNA
Cleaved Primary mRNA
5’
5’
Unintended mRNA Target
3’
Cleaved Unintended mRNA
• A-siRNA’s non-functional sense results in
elimination of “Off-Target” suppression of genes
complimentary to sense RNA.
• 100% processing effiency to target mRNA due
to asymmetric structure and end thermodynamics.
Oligoengine
• siRNA results in “Off-Target” suppression of
genes partial complimentary to sense RNA.
• Overall molecule is double-processed
(forward/reverse) resulting in decreased efficiency.
• More stable in Cytosol and Serum than dsRNA.
CORPORATE OFFICE
1409 42nd Ave. E.
Seattle, WA 98112
LABORATORY
Seattle, WA
Phone:
Fax:
Toll-free:
(206) 254-0200
(206) 254.0300
(800) 516.5446
A-siRNA
Asymmetric siRNA Duplex
TM
Invivo use of Asymmetric siRNA Duplexes.
The A-siRNADuplex is more effective in-vivo than siRNA due to it’s stability and
processing efficiency. A direct comparison was performed against siRNA for serum
stability and invivo efficacy by a third party.
siRNA
Serum
Stability
0
5
10
15
30
A-siRNA Duplex
60
90
0
Minutes in Serum
5
10
15
30
60
90
Hydrodynamic tail vein injection of 10mg of siRNA or A-siRNA against the Fas
receptor mRNA.
In-vivo against fas
Results from studies at an independent,
third-party research institution indicate
that intravenous injection of A-siRNA
Duplexes targeting the Fas gene in
mouse hepatocytes reduces Fas mRNA
levels and expression of Fas protein in
the liver. The goal is to demonstrate
the potential therapeutic value of the
A-siRNA Duplex; in this case, in
preventing and treating acute and
chronic liver injury induced by viral
and autoimmune hepatitis, alcoholic
liver disease, acute and chronic liver
failure and rejection of liver transplants.
Introduce FAS target to 30 mice
10 w/siRNA
10 w/A-siRNA
10 w/Saline
1. Compare Knockdown efficacy between
siRNA and Asymmetric siRNA Duplexes.
2. Profile liver tissue and compare potential
off-target activity of siRNA and Asymmetric
siRNA Duplexes.
Sacrifice at 48 hours.
In-vivo results
NS
Analysis by real time PCR of Fas mRNA.
1.2
* p=0.01
Level of mRNA
1
.8
.6
.4
.2
0
siRNA 48h
A-siRNA 48h
saline 48h
Oligoengine
CORPORATE OFFICE
1409 42nd Ave. E.
Seattle, WA 98112
LABORATORY
Seattle, WA
Phone:
Fax:
Toll-free:
(206) 254-0200
(206) 254.0300
(800) 516.5446
A-siRNA
Asymmetric siRNA Duplex
TM
Asymmetric siRNA Duplex Summary.
Summary
• More Potent
• More Specfic
• More Stable
Prices start at $399.00 per 40 nm duplex. Please visit http://www.oligoengine.com/oe3
to design and order your A-siRNA Duplex or call Customer Service.
Intellectual Property The A-siRNA Duplex was developed and is supplied exclusively by
OligoEngine’s parent company, Halo-Bio RNAi Therapeutics.
Like small interfering RNA, the A-siRNA Duplex consists of two short polynucleotides,
custom synthesized to target a specific gene sequence, and annealed in a duplex
molecule. And like siRNA, the A-siRNA Duplex has been demonstrated as a potent
RNAi trigger.
However, important differences in structure, chemistry and configuration exist
between the A-siRNA Duplex and “standard” siRNA molecules. As such, the AsiRNA Duplex is the subject of a patent application filed by Oligoengine scientists
with the United States Patent and Trademark Office (patent pending).
Licensing
Licensing of A-siRNA for Target Validation, Drug Discovery, and
Therapeutic Applications
The A-siRNA Duplex has utility across all stages of the gene-to-drug pathway.
Commercial parties can obtain a license of the A-siRNA technology for the purposes
of performing target validation, drug discovery and development, or therapeutics
applications. In addition, any products, including cell lines, transgenic animals and
therapeutic compounds developed using the A-siRNA Duplex will be considered as
licensed products, upon which Halo-Bio will seek to support such uses by licensing.
Terms of a commercial license are available for various uses and geographies. For
specific licensing information, please contact an OligoEngine sales manager or HaloBio RNAi Therapeutics directly:
http://www.halo-bio.com
Oligoengine
CORPORATE OFFICE
1409 42nd Ave. E.
Seattle, WA 98112
LABORATORY
Seattle, WA
Phone:
Fax:
Toll-free:
(206) 254-0200
(206) 254.0300
(800) 516.5446
A-siRNA
Asymmetric siRNA Duplex
TM
EXCLUSIVE:
Asymmetric siRNA Duplex
TM
fax-back
quote form
Advanced RNAi Kit
Order the A-siRNA Duplex to achieve greater
suppression, specificity and stability over typical siRNA.
Fax it back, we’ll take care of the rest!
Fax to 206.254.0300
KIT CONTENTS:
• 40 nMole of A-siRNA Duplex
IDENTITY:
• Dilution Buffer
NAME:
• Optional positive or negative controls
PHONE:
EMAIL:
GENE OR SEQUENCE:
No Additional Charge for Design. Results guaranteed
to acheive greater than 70% suppression.
GENE(S):
N19 SEQUENCE(S):
KIT OPTIONS:
ADD CUSTOM NEGATIVE CONTROL $199
5’ LABEL 20 nM of Si2 Duplex $199
6-FAM
HEX
TET
TAMRA
POSITIVE/NEGATIVE CONTROLS
starting at
$399.00
5 nmoles anti-GFP duplex $99
5 nmoles Mamm-X Scramble duplex $99
A-siRNA Duplex
For more information and licensing terms:
1.800.516.5446
customerservice@oligoengine.com
Oligoengine
CORPORATE OFFICE
1409 42nd Ave. E.
Seattle, WA 98112
LABORATORY
Seattle, WA
Phone:
Fax:
Toll-free:
(206) 254-0200
(206) 254.0300
(800) 516.5446
A-siRNA
© 2010 Halo-Bio RNAi Therapeutics, Inc. All Rights Reserved.
Asymmetric siRNA Duplex
TM