Catalog #: AM8045

m7(3'-O-methyl)-
G(5')ppp(5')G (ARCA)
Store below –70°C.
Do not store in a frost-free freezer.

Catalog #: AM8045
Product Description: An Anti-Reverse Cap Analog (ARCA) designed for synthesis of capped RNAs that are 100% functional in in vitro
protein synthesis.
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Amount: 10 A255 Units (10 A255 = 44.4 x 10 mol = 0.392 mg)

Volume: 11.1 μL
Concentration: 40 mM
Molecular Weight: 884 (Disodium salt)
Structure:

Storage Conditions: Store at or below –70°C. Avoid multiple freeze-thaw cycles. Aliquots of the product may be stored short-term at
–20°C. Do not store in a frost-free freezer.
Storage Buffer: Nuclease-free Water

USER INFORMATION

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General Information: A modified nucleotide structure (N -methyl guanosine; m G), known as "cap," is present on the 5' end of most
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naturally occurring eukaryotic mRNAs and many viral RNAs. In vivo, the m G cap structure is added enzymatically to
mRNA produced by RNA polymerase II. During in vitro transcription reactions catalyzed by viral RNA polymerases,
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m G(5')pppG cap analog is incorporated as the first nucleotide for the synthesis of 5' capped RNAs. However,
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standard cap analog can be incorporated into the RNA in the forward [m G(5')pppG(pN)] or the reverse orientation
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[G(5')pppm G(pN)] and leads to synthesis of two forms of isomeric RNAs [1]. RNAs capped with reverse orientation
5' caps are not efficiently translated.

A novel cap analog, Anti-Reverse Cap Analog (ARCA), has been described in two publications [2, 3]. In ARCA, one
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of the 3' OH groups (closer m G) is eliminated from the cap analog and is substituted with -OCH3. This modification
forces RNA polymerases to initiate transcription with the remaining -OH group in G and thus synthesize RNA
transcripts capped exclusively in the correct orientation. Substitution of the traditional cap analog with ARCA allows
for synthesis of capped RNAs that are 100% functional, in contrast to transcription reactions using traditional cap
analogs where only half of the cap analog is incorporated in the correct orientation [1]. Capped mRNAs can be used
for protein synthesis studies in reticulocyte lysate, wheat germ lysate, and other in vitro systems, or can be
microinjected or transfected into cells or organisms for in vivo studies. They can also be used in RNA splicing and
stability studies.
Applications: For convenience, ARCA is supplied at a concentration of 40 mM in water. If it is necessary to concentrate it, ARCA
®
may be lyophilized or dried in a SpeedVac Concentrator without heat.

In vitro transcripts that will be microinjected into oocytes or other cells, used for transfection experiments, or used in
in vitro splicing reactions, should be capped, because uncapped mRNAs are rapidly degraded after microinjection or
transfection into cells. However, there are additional factors that should be considered:
● In vitro transcription reactions that contain high levels of cap analog produce significantly lower yields of
transcript.
● Unincorporated cap analog is a potent inhibitor of protein synthesis initiation.

Ambion, Inc. • An Applied Biosystems Business • +1 800-888-8804 • www.ambion.com • e-mail: techserv@ambion.com

Catalog #: AM8045 1/7/2008

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 The standard strategy used to synthesize capped transcripts is to reduce the level of GTP to 10% of the normal
concentration and replace the remaining 90% with cap analog [4]. This results in a high proportion of the transcripts
being capped. Unfortunately, it also significantly decreases the yield of the transcription reaction, often to 20% or
lower. To conserve cap analog and increase the yield of the transcription reaction, many scientists use a lower ratio
of cap analog to GTP. A cap analog:GTP ratio of 4:1 is frequently used, but ratios as low as 1:1 are also used. It is
likely, however, that the fraction of capped mRNA molecules decreases as the ratio of cap analog to GTP decreases.
Thus, we recommend using a 4:1 ratio of ARCA to GTP as a reasonable compromise, unless it is essential that a
very high fraction of the transcripts be capped.
® ®
ARCA can be added to Ambion MEGAscript High Yield Transcription Kits (Cat #AM1330, AM1334, AM1338) to
efficiently synthesize large amounts of ARCA-capped RNA. The kit protocol and manual are available at
www.ambion.com/catalog/CatNum.php?1334. For additional information on the procedure and protocol, please
contact the Ambion Technical Services Department (1-800-888-8804, option 2 or TechServ@ambion.com).

References: 1. Pasquinelli AE, Dahlberg JE, Lund E (1995) Reverse 5' caps in RNAs made in vitro by phage RNA
polymerases. RNA 1(9): 957–967.
2. Stepinski J, Waddell C, Stolarski R, Darzynkiewicz E, Rhoads RE (2001) Synthesis and properties of mRNAs
containing the novel "anti-reverse" cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl(3'deoxy)GpppG.
RNA 7: 1486–1495.
3. Peng Z-H, Sharma V, Singleton SF, and Gershon PD (2002) Synthesis and applications of chain-terminating
dinucleotide mRNA cap analog. Organic Letters 4: 161–164.
4. Krieg PA, Melton DA (1987) In vitro RNA synthesis with SP6 RNA polymerase. Methods Enzymol 155:
397–415.

QUALITY CONTROL

Nonspecific Endonuclease Meets or exceeds specification when a sample is incubated for 14–16 hr with 300 ng supercoiled plasmid DNA and
Activity: analyzed by agarose gel electrophoresis.
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Exonuclease Activity: Meets or exceeds specification when a sample is incubated for 14–16 hr with 40 ng P-labeled Sau3A fragments of
pUC19 and analyzed by PAGE.
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RNase Activity: Meets or exceeds specification when a sample is incubated for 14–16 hr with 25 ng P-labeled RNA and analyzed
by PAGE.
Functional Testing: ARCA is analyzed for purity by HPLC and is shown to be ≥95% pure. Capping efficiency is tested using the
® ®
MAXIscript Kit (Cat #AM1312) and a gel shift assay. ARCA is tested in the MEGAscript Kit (Cat #AM1334) for yield
of capped RNA.

OTHER INFORMATION

Material Safety Data Sheets:
Warranty and Liability:
Material Safety Data Sheets (MSDSs) can be printed or downloaded from product-specific links on our website at the following
address: www.ambion.com/techlib/msds. Alternatively, e-mail your request to MSDS_Inquiry_CCRM@appliedbiosystems.com.
Specify the catalog or part number(s) of the product(s), and we will e-mail the associated MSDSs unless you specify a preference for
fax delivery. For customers without access to the internet or fax, our technical service department can fulfill MSDS requests placed by
telephone or postal mail. (Requests for postal delivery require 1–2 weeks for processing.)
For research use only. Not for use in diagnostic procedures.

Applied Biosystems is committed to delivering superior product quality and performance, supported by industry-leading global service
and technical support teams. Warranty information for the accompanying consumable product is available at
www.ambion.com/info/warranty in "Limited Warranty for Consumables," which is subject to the exclusions, conditions, exceptions,
and limitations set forth under the caption “EXCLUSIONS, CONDITIONS, EXCEPTIONS, AND LIMITATIONS” in the full warranty
statement. Please contact Applied Biosystems if you have any questions about our warranties or would like information about
post-warranty support.
Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that
may appear in this document.

Applied Biosystems disclaims all warranties with respect to this document, expressed or implied, including but not limited to those of
merchantability or fitness for a particular purpose. In no event shall Applied Biosystems be liable, whether in contract, tort, warranty,
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© 2007 Ambion, Inc. All rights reserved. 4386601A

Ambion, Inc. • An Applied Biosystems Business • +1 800-888-8804 • www.ambion.com • e-mail: techserv@ambion.com

Catalog #: AM8045 1/7/2008

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