feature article

Minding your caps and tails – considerations for functional

mRNA synthesis
Applications of synthetic mRNA have grown and become considerably diversified in recent years. Examples include the generation of
pluripotent stem cells (1-3), vaccines and therapeutics (4), and CRISPR/Cas9 genome editing applications (5-7). The basic requirements
for a functional mRNA – a 7-methylguanylate cap at the 5´ end and a poly(A) tail at the 3´ end – must be added in order to obtain efficient
translation by eukaryotic cells. Additional considerations can include the incorporation of modified bases, modified cap structures and
polyadenylation strategies. Strategies for in vitro synthesis of mRNA may also vary according to the desired scale of synthesis. This article
discusses options for selection of reagents and the extent to which they influence synthesized mRNA functionality.

Breton Hornblower, Ph.D., G. Brett Robb, Ph.D. Figure 2. Methods for generating transcription templates
and George Tzertzinis, Ph.D., New England
Biolabs, Inc. A. PCR-Based Strategy B. Blunt Versus Type IIS Enzyme-Based Strategies

Before translation in eukaryotic organisms, na- BspQI

5´ 3´ 5´... N N N N N N N N N N ...3´ 5´... N N N N N G A A G A GC ...3´
scent mRNA (pre-mRNA) receives two significant 3´ 5´ 3´... N N N N N N N N N N ...5´ 3´... N N N N N C T T C T CG ...5´
modifications in addition to splicing. During syn- Target DNA
thesis, a 7-methylguanylate structure, also known
PCR
as a “cap”, is added to the 5´ end of the pre-
mRNA, via 5´ → 5´ triphosphate linkage. This 5´ 3´ Target
cap protects the mature mRNA from degradation, 3´ 5´ DNA
5´
Primer
and also serves a role in nuclear export and trans- Primer
lation initiation. The second modification is the 3´
3´ 5´
addition of approximately 200 adenylate nucleo-
tides (a poly(A) tail) to the 3´ end of pre-mRNA Blunt Digestion
digestion with BspQI
by E. coli Poly(A) Polymerase. Polyadenylation 5´ 3´
is coupled to transcription termination, export 3´ 5´
of mRNA from the nucleus, and, like the cap, 5´... 3´ 5´... 3´
5´ 3´ 3´... 5´ 3´... 5´
formation of the translation initiation complex. 3´ 5´
The mature mRNA forms a circular structure by
bridging the cap to the poly(A) tail via the cap- RNA
binding protein eIF4E (eukaryotic initiation fac- polymerase
tor 4E) and the poly-(A) binding protein, both of promoter
which interact with eIF4G (eukaryotic initiation 5´ 3´
factor 4G). (Figure 1, (8)) 3´ 5´
DNA template
RNA can be efficiently synthesized in vitro with
prokaryotic phage polymerases, such as T7, T3 (A) PCR can be used to amplify target DNA prior to transcription. A promoter can be introduced via the upstream primer.
and SP6. The cap and poly(A) tail structures (B) When using plasmid DNA as a template, linearize with an enzyme that produces blunt or 5´-overhanging ends. Using a type IIS
characteristic of mature mRNA can be added restriction enzyme (e.g., BspQI) allows RNA synthesis with no additional 3´-nucleotide sequence from the restriction site.

during or after the synthesis by enzymatic

reactions with capping enzymes and Poly(A) ence the ease of experimental setup and yield of PCR allows conversion of any DNA fragment
Polymerase, respectively. the final mRNA product. These are discussed in to a transcription template by appending the T7
the following sections. promoter to the forward primer (Fig 2A). Ad-
There are several factors to consider when plan-
ditionally, poly(d)T-tailed reverse primers can be
ning for in vitro mRNA synthesis that will influ- DNA template used in PCR to generate transcription templates
The DNA template provides the sequence to be with A-tails. This obviates the need for a separate
transcribed downstream of an RNA polymerase polyadenylation step following transcription. Re-
Figure 1. Translation initiation complex. promoter. There are two strategies for generating peated amplifications should, however, be avoided
transcription templates: PCR amplification and to prevent PCR-generated point mutations. Am-
3´ UTR
AAAAAAAAAAAAAA linearization of plasmid with a restriction enzyme plification using PCR enzymes with the highest
Poly(A) Binding Proteins (Figure 2). Which one to choose will depend on possible fidelity, such as Q5® High-Fidelity DNA
(PABPs)
the downstream application. In general, if mul- Polymerase (NEB # M0491), reduces the likeli-
CDS tiple sequences are to be made and transcribed in hood of introducing such mutations.
parallel, PCR amplification is recommended as it
generates many templates quickly. On the other The quality of the PCR reaction can be assessed
eIF4E Cap hand, if large amounts of one or a few templates by running a small amount on an agarose gel, and
40S 5´ UTR
are required, plasmid DNA is recommended, DNA should be purified before in vitro transcrip-
because of the relative ease of producing large tion using a spin column or magnetic beads (e.g.,
The mature RNA forms a circular structure connected by protein quantities of high quality, fully characterized AMPure® beads). Multiple PCR reactions can be
complexes that bind the cap structure and poly(A) tail. plasmids. purified and combined to generate a DNA stock
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feature article

solution that can be stored at -20°C, and used as restriction enzyme, followed by purification using Figure 4. RNA yields from transcriptional
needed for in vitro transcription. a spin column or phenol extraction/ethanol pre- capping reactions
cipitation. Although linearization of plasmid in-
Plasmid templates are convenient if the template
volves multiple steps, the process is easier to scale 150 GLuc 150
sequence already exists in a eukaryotic expres-
for the generation of large amounts of template
sion vector also containing the T7 promoter (e.g.,
for multiple transcription reactions.
pcDNA vector series). These templates include 100 100
5´- and 3´-untranslated regions (UTR), which are
In vitro transcription

µg RNA

µg RNA
important for the expression characteristics of the
mRNA. There are two options for the in vitro transcription
50 50
reaction depending on the capping strategy cho-
Plasmid DNA should be purified and linearized sen: standard synthesis with enzyme-based cap-
downstream of the desired sequence, preferably ping following the transcription reaction (post- 0 0
with a restriction enzyme that leaves blunt or 5´ transcriptional capping) or incorporation of a cap Enzyme ARCA 7mG Enzyme A
based Co-transcriptional based C
overhangs at the 3´ end of the template. These analog during transcription (co-transcriptional
are favorable for proper run-off transcription by capping) (Fig. 3). Method selection
150 will depend 150
GLuc CLuc
T7 RNA Polymerase, while 3´ overhangs may re- on the scale of mRNA synthesis required and
sult in unwanted transcription products. To avoid number of templates to be transcribed.
adding extra nucleotides from the restriction site 100 100
to the RNA sequence, a Type IIS restriction en- Transcription for enzyme-based capping

µg RNA

µg RNA
zyme can be used (e.g., BspQI, NEB #R0712), (post-transcriptional capping)
which positions the recognition sequence outside Standard RNA synthesis reactions50produce the 50
of the transcribed sequence (Figure 2B). The plas- highest yield of RNA transcript (typically ≥100
mid DNA should be completely digested with the µg per 20 µl in a 1 hr reaction using the HiScribe
0 0
Enzyme ARCA 7mG Enzyme ARCA 7mG
based Co-transcriptional based Co-transcriptional
Figure 3. In vitro transcription options based upon capping strategy
Reactions were set up according to recommended conditions for
Enzyme-Based mRNA Capping Co-Transcriptional mRNA Capping two templates: Gaussia luciferase (GLuc) and Cypridina luciferase
(CLuc). The RNA was quantified spectrophotometrically after
5´ 3´ purification with spin columns.
3´ 5´
RNA polymerase DNA template
promoter
Quick T7 High Yield RNA Synthesis Kit, NEB
#E2050S). Transcription reactions are highly
Phosphate
Co-transcriptional scalable, and can be performed using an all-
5´ P P P 3´ capping with ARCA inclusive kit (e.g., HiScribe kits), or individual
RNA transcript reagents. More information on the HiScribe kits
m7G P P P
can be found on page 7.
P P P G GTP
Capping with Vaccinia
SAM Following transcription, the RNA is treated with
Capping System
DNase I to remove the DNA template, and puri-
fied using an appropriate column, kit or magnetic
5´ m7G P P P 3´ 5´ m7G P P P 3´ beads, prior to capping. This method produces
Cap-0 mRNA Cap-0 mRNA high yields of RNA with 5´-triphosphate termini
+ that must be converted to cap structures. In the
absence of template-encoded poly(A) tails, tran-
5´ P P P 3´
scripts produced using this method bear 3´ termi-
SAM Cap-1 methylation Uncapped RNA transcript
using mRNA Cap ni that also must be polyadenylated in a separate
2´-O-Methyltransferase enzymatic step, as described below in “Post-tran-
SAM Cap-1 methylation
using mRNA Cap scriptional capping and Cap-1 methylation”.
2´-O-Methyltransferase

Transcription with co-transcriptional capping

Methylation Methylation
OCH3 in Cap-1 OCH3 in Cap-1 With co-transcriptional capping, a cap analog is
5´ m7G P P P 3´ 5´ m7G P P P 3´ introduced into the transcription reaction, along
Cap-1 mRNA Cap-1 mRNA
with the four standard nucleotide triphosphates,
in an optimized ratio of cap analog to GTP 4:1.
This allows initiation of the transcript with the
Enzyme-based capping (left) is performed after in vitro transcription using 5´-triphosphate RNA, GTP, and S-adenosyl-methionine (SAM).
Cap 0 mRNA can be converted to cap 1 mRNA using mRNA cap 2´-O-methyltransferase (MTase) and SAM in a subsequent or concurrent
cap structure in a large proportion of the synthe-
reaction. The methyl group transferred by the MTase to the 2´-O of the first nucleotide of the transcript is indicated in red. Conversion of sized RNA molecules. This approach produces
~100% of 5´-triphosphorylated transcripts to capped mRNA is routinely achievable using enzyme-based capping. a mixture of transcripts, of which ~80% are
Co-transcriptional capping (right) uses an mRNA cap analog (e.g., ARCA; anti-reverse cap analog), shown in yellow, in the transcription capped, and the remainder have 5´-triphosphate
reaction. The cap analog is incorporated as the first nucleotide of the transcript. ARCA contains an additional 3´-O-methyl group on the
ends. Decreased overall yield of RNA products
7-methylguanosine to ensure incorporation in the correct orientation. The 3´-O-methyl modification does not occur in natural mRNA caps.
Compared to reactions not containing cap analog, transcription yields are lower. ARCA-capped mRNA can be converted to cap 1 mRNA results from the lower concentration of GTP in
using mRNA cap 2´-O-MTase and SAM in a subsequent reaction. the reaction (Fig. 4).
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feature article

Figure 5. Structure of the anti-reverse cap analog, ARCA m7G-cap structures. The Vaccinia Capping Sys-
tem (NEB #M2080) is comprised of three enzy-
matic activities (RNA triphosphatase, guanylyl-
transferase, guanine N7-methyltransferase) that
are necessary for the formation of the complete
Cap-0 structure, m7Gppp5´N, using GTP and
the methyl donor S-adenosylmethionine. As an
added option, the inclusion of the mRNA Cap
2´ O-Methyltransferase (NEB #M0366) in the
same reaction results in formation of the Cap-1
The 3´ position of the 7-methylated G is blocked by a methyl group.
structure, which is a natural modification in many
eukaryotic mRNAs. This enzyme-based cap-
ping approach results in the highest proportion
There are several cap analogs used in co-tran- tionally capped mRNAs, as described above. of capped message, and it is easily scalable. The
scriptional RNA capping. The most common Alternatively, the HiScribe T7 Quick RNA Syn- resulting capped RNA can be further modified by
are the standard 7-methyl guanosine (m7G) cap thesis Kit may be used to produce transcripts for poly(A) addition before final purification.
analog and anti-reverse cap analog (ARCA), post-transcriptional capping (see below).
also known as 3´ O-me 7-meGpppG cap analog
(Fig. 5). ARCA is methylated at the 3´ position Post-transcriptional capping and
of the m7G, preventing RNA elongation by Cap-1 methylation
phosphodiester bond formation at this position. Post-transcriptional capping is often performed
Thus, transcripts synthesized using ARCA con- using the mRNA capping system from Vac-
tain 5´-m7G cap structures in the correct orienta- cinia virus. This enzyme complex converts the
tion, with the 7-methylated G as the terminal 5´-triphosphate ends of in vitro transcripts to the
residue. In contrast, the m7G cap analog can be
incorporated in either the correct or the reverse
orientation. Analysis of capped RNA function in transfected mammalian cells

HiScribe T7 ARCA mRNA Synthesis Kits (NEB# A.

E2060 and E2065) contain reagents, including
an optimized mix of ARCA and NTPs for stream-
lined reaction setup for synthesis of co-transcrip-
tionally capped RNAs.
Make mRNA Transfect cells Harvest media Read light output
Transcription with complete substitution with
Schematic representation of reporter mRNA transfection workflow.
modified nucleotides
RNA synthesis can be carried out with a mixture
of modified nucleotides in place of the regular The effect of capping can be studied by delivering the mRNA to cultured mammalian cells and monitoring its translation.
Using RNA encoding secreted luciferases (e.g., Cypridina luciferase, CLuc) the translation can be monitored by assaying its
mixture of A, G, C and U triphosphates. For ex-
activity in the cell culture medium (Fig. A).
pression applications, the modified nucleotides of
CLuc mRNA was synthesized and capped post-transcriptionally (Cap 0 or Cap 1) or co-transcriptionally (as described
choice are the naturally occurring 5´-methylcyti-
above) using standard (7mG) or anti-reverse cap analog (ARCA). For consistency, the mRNAs were prepared from templates
dine and/or pseudouridine in the place of C and encoding poly-A tails of the same length.
U, respectively. These have been demonstrated
After capping, the mRNA was purified using magnetic beads and quantified before transfection into U2OS cells using
to confer desirable properties to the mRNA, such the TransIT® mRNA transfection reagent following the manufacturer’s protocol. CLuc activity was measured 16 hrs after
as higher expression levels and avoidance of transfection using the BioLux® Cypridina Luciferase Assay Kit (NEB #E3309).
unwanted side effects in the key applications of Virtually no luciferase reporter activity was observed in conditions where uncapped RNA was transfected (Fig. B). In contrast,
protein replacement and stem-cell differentiation robust activity was detected from cells transfected with RNA capped using the methods described above. As anticipated,
(1). It is important to note that nucleotide choice lower activity was observed from cells transfected with mRNA capped using the 7mG cap analog as compared to ARCA-
capped mRNA.
can influence the overall yield of mRNA synthesis
reactions.
B. 2.0x107
Fully substituted RNA synthesis can be achieved
using the HiScribe T7 High-Yield RNA Syn-
1.5x107
thesis Kit (NEB# E2040) in conjunction with
NTPs with the desired modification. Transcripts
RLU

1.0x107
made with complete replacement of one or more
nucleotides may be post-transcriptionally capped
5.0x106
(see next section), or may be co-transcriptionally
capped by including ARCA or another cap ana-
0.0
log, as described previously.
Cap 0 Cap 1 7mG ARCA Uncapped
Cap Structure
If partial replacement of nucleotides is desired,
the HiScribe T7 ARCA mRNA Synthesis Kits
Expression of Cypridina luciferase (CLuc) after capping using different methods. High activity from all capped RNAs is
(NEB# E2060 and E2065), may be used with observed.
added modified NTPs, to produce co-transcrip-
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feature article

A-tailing using E. coli Poly(A) The importance of the A-tail is demonstrated by for the enzymatic tailing of co-transcriptionally
Polymerase transfection of untailed vs. tailed mRNA. When capped RNA. For mRNA synthesis from templates
luciferase activity from cells transfected with equi- with encoded poly(A) tails, the HiScribe T7 ARCA
The poly(A) tail confers stability to the mRNA
molar amounts of tailed or untailed mRNAs were mRNA Synthesis Kit (NEB# E2065) provides
and enhances translation efficiency. The poly(A)
compared, a significant enhancement of transla- and optimized formulation for co-transcriptionally
tail can be encoded in the DNA template by us-
tion efficiency was evident (Fig. 6). Increasing the capped transcripts.
ing an appropriately tailed PCR primer, or it
length of poly(A) tails did not markedly further
can be added to the PCR-amplified template by Summary
enhance reporter activity.
enzymatic treatment of RNA with E. coli Poly(A)
In summary, when choosing the right workflow
Polymerase (NEB #M0276). The lengths of HiScribe T7 ARCA mRNA Synthesis Kit (with
for your functional mRNA synthesis needs, you
the added tails can be adjusted by titrating the tailing) (NEB# E2060) includes E. coli Poly(A)
must balance your experimental requirements for
Poly(A) Polymerase in the reaction (Fig. 6). Polymerase, and enables a streamlined workflow
the mRNA (e.g., internal modified nucleotides)
with scalability (i.e., ease-of-reaction setup vs.
Figure 6. Analysis of capped and polyadenylated RNA yield of final product).

A. B. In general, co-transcriptional capping of mRNA

5.0x107 with template encoded poly(A) tails or post-tran-
scriptional addition of poly(A) tail is recommended
4.0x107
for most applications. This approach, using the
HiScribe T7 ARCA mRNA Synthesis Kits (NEB#
3.0x107
E2060 and E2065), enables the quick and stream-
RLU

2.0x107 lined production of one or many transcripts with

typical yields of ~20 µg per reaction, totaling
1.0x107 ~400 µg per kit.
0.0 Post-transcriptional mRNA capping with Vac-
0 10 20 30
PAP units
cinia Capping System is well suited to larger scale
synthesis of one or a few mRNAs, and is readily
scalable to produce gram-scale quantities and be-
yond. Reagents for in vitro synthesis of mRNA are
A. Agilent Bioanalyzer® analysis of capped and polyadenylated RNA. Longer tails are produced by increasing the enzyme
concentration in the reaction. Calculated A-tail lengths are indicated over each lane. Lanes: L: size marker,1: No poly-A tail, 2: 5 available in kit form and as separate components
units, 3 :15 units, 4 : 25 units of E. coli Poly(A) Polymerase per 10 µg CLuc RNA in a 50 µL reaction. to enable research and large-scale production.
B. Effect of enzymatic A-tailing on the luciferase reporter activity of CLuc mRNA
Products available from NEB for each step of the
functional mRNA synthesis workflow, from tem-
plate construction to tailing, are shown to the left.

Products from NEB are available for each step of the RNA Synthesis Product Workflow References:
1. Warren, L., et al. (2010) Cell Stem Cell, 7, 618-630.
2. Angel, M. and Yanik, M.F. (2010) PLoS One, 5:e11756.
Template In vitro RNA Poly(A) 3. Yakubov, E., et al. (2010) Biochem. Biophys. Res. Commun. 394, 189.
Generation Transcription Capping Tailing 4. Geall, A.J., et al. (2012) P roc. Natl. Acad. Sci. USA, 109, 14604-14609.
5. Ma, Y., et al. (2014) PLoS One, 9:e89413.
6. Ota, S., et al. (2014) Genes Cells, 19, 555-564.
7. Andrew R. B., et al. (2013) Cell Rep. 4, 220–228.
Q5® High-Fidelity HiScribe™ T7 ARCA mRNA Synthesis Kit (with tailing) 8. Wells, S.E., et al. (1998) Molecular Cell 2, 135–140.

DNA Polymerase
HiScribe T7 ARCA mRNA Synthesis Kit E. coli Poly(A) Polymerase
dNTP solution mixes
HiScribe T7 High Vaccinia
Type IIs Yield RNA Synthesis Kit Capping System
restriction enzymes
& cloning reagents HiScribe T7 Quick High mRNA Cap 2´-O-
Yield RNA Synthesis Kit Methyltransferase

T3, T7 and SP6 ARCA and other

RNA Polymerases mRNA cap analogs

Companion Products
RNase inhibitors

Pyrophosphatases

DNase I

NTPs

www.neb-online.de
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