Synthesis of biotin labelled cap analogue - Incorporable into mRNA transcripts and promoting cap-dependent translation

Article abstract of DOI:10.1039/c2ob26060c

Analogues of the eukaryotic messenger RNA 5′ end (m⁷G cap) are useful tools for studying mRNA fate and serve as reagents for in vitro preparation of 5′ capped mRNAs. We designed a biotin-labeled dinucleotide cap analogue that can be incorporated into transcripts to produce 5′-capped and biotinylated mRNAs which retain their biological functionality and may be employed for biotin-(strept)avidin technologies.

Full text of DOI:10.1039/c2ob26060c

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Volume 10 | Number 43 | 21 November 2012 | Pages 8553–8740
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Jacek Jemielity et al.
Synthesis of biotin labelled cap analogue – incorporable into mRNA transcripts
and promoting cap-dependent translation

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COMMUNICATION
Synthesis of biotin labelled cap analogue – incorporable into mRNA
transcripts and promoting cap-dependent translation†
Jacek Jemielity,* Maciej Lukaszewicz, Joanna Kowalska, Jakub Czarnecki, Joanna Zuberek and
Edward Darzynkiewicz
Received 1st June 2012, Accepted 10th July 2012
DOI: 10.1039/c2ob26060c
Analogues of the eukaryotic messenger RNA 5′ end (m7G cap)
horseradish peroxidase or fluorescent dyes, as well as for label-
ling molecules for imaging or delivery of therapeutics.11–13
are useful tools for studying mRNA fate and serve as
Different approaches have been developed for nucleic acid label-
ling with biotin. RNA has been labelled both at the intrastrand
positions and at the 5′ or 3′ ends.14–20 Such labelled RNAs are
useful for many applications; however, since the cap is involved
in crucial processes of mRNA metabolism, particularly initiation
of translation, their use for studying eukaryotic mRNA function
may be limited. In the case of our studies we aimed at develop-
ing a biotin-labelled analogue of the mRNA cap that would fulfil
the following three conditions: (1) specifically interacts with
avidin/streptavidin. (2) enables synthesis of 5′-biotinylated
capped full-length mRNA by transcription in vitro, i.e. serves as
a substrate for bacteriophage RNA polymerases during initiation
of transcription. (3) bears the biotin label at a position that intro-
duced the smallest possible disturbance of cap–eIF4E inter-
action, thus enabling efficient translation of the biotinylated
mRNA.
reagents for in vitro preparation of 5′ capped mRNAs. We
designed a biotin-labeled dinucleotide cap analogue that can
be incorporated into transcripts to produce 5′-capped and bio-
tinylated mRNAs which retain their biological functionality
and may be employed for biotin–(strept)avidin technologies.
All eukaryotic messenger RNAs possess a cap at their 5′ end,
consisting of 7-methylguanosine (m7G) linked by a 5′,5′-triphos-
phate bridge to the first transcribed nucleotide.1 This structure,
distinct from typical nucleic acids, serves as an intrinsic mRNA
5′ end tag, which enables selective binding to proteins involved
in mRNA translation and turnover. These include cytoplasmic
translation initiation factor 4E (eIF4E), the nuclear cap binding
complex (CBC) or decapping enzymes (Dcp2, hNUDT16,
DcpS).2–9
Not surprisingly then, synthetic cap analogues have been used
for studying numerous mRNA-related processes, e.g. translation,
translational repression or mRNA decay pathways.10 In order to
further facilitate studies on gene expression by means of cap-
derived molecular tools, in this work we propose a simple
method for obtaining mRNA molecules site-specifically biotiny-
lated within the 5′ cap. We describe an efficient chemical syn-
thesis of a biotin-labelled dinucleotide cap analogue, which can
be incorporated into transcripts by enzymatic transcription
in vitro. We also demonstrate that biotinylated transcripts
obtained in this manner undergo cap-dependent translation and
are bound by streptavidin. Hence, these transcripts retain their
biological activity, coincidently being applicable for biotin–
(strept)avidin interaction based experiments.
Such biotin-labelled mRNAs could be useful e.g. for deter-
mining cellular localization and following the fate of capped
mRNAs, for pull-down assays, ELISA tests, western blots and
other immuno-analytical methods.
To enable attachment of biotin through its carboxylic group,
we designed a dinucleotide cap analogue 1 bearing an aliphatic
amino group at the 2′-position of 7-methylguanosine. The ana-
logue was converted into its biotinylated derivative 2 by coup-
ling with NHS-activated biotin (Fig. 1). The biotin NHS-ester
was conveniently generated in situ using O-(N-succinimidyl)-
1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU).‡ The
coupling reaction of 1 with NHS-biotin was highly selective and
efficient. The HPLC analysis revealed a complete conversion of
1 into 2 (Fig. 2) within 60 min. No by-products derived from the
coupling of nucleobase exocyclic amino groups or ribose
hydroxyl groups have been detected. After RP-HPLC purifi-
cation the triammonium salt of compound 2 was isolated with a
60% yield.
The biotin tag is commonly used for purification, detection
and visualization of proteins and nucleic acids via anti-biotin
antibodies or avidin/streptavidin tagged detectors, such as
The synthetic route for cap analogue 1 is depicted in Fig. 3.
The synthesis has been designed to minimize the number of
steps employing 2′-amino-2′-deoxyguanosine. It has been found
that the 2′-amino group does not require protection, neither at the
5′-phosphorylation nor during the N7-methylation step, which
made the synthesis relatively straightforward. The procedure
Division of Biophysics, Institute of Experimental Physics, Faculty of
Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw,
Poland. E-mail: jacekj@biogeo.uw.edu.pl
†Electronic supplementary information (ESI) available: Fig. S1–S6,
Tables S1 and S2, detailed experimental procedures, HRMS, 1H NMR
and 31P NMR spectra of compounds
10.1039/c2ob26060c
1 and 2. See DOI:
8570 | Org. Biomol. Chem., 2012, 10, 8570–8574
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Fig. 3 Synthesis of cap analogue 1. Reaction conditions: (i) 1. POCl3
(3 equiv.), (MeO)3PO, 0 °C, 8 h; 2. H2O, NaHCO3; (ii) CH3I (8 equiv.),
DMSO,
4 h; (iii) 1. Imidazole (10 equiv.), 2,2′-dithiodipyridine
(3 equiv.), triphenylphosphine (3 equiv.), TEA, DMF, 8 h; 2. NaClO4,
acetone; (iv) 4 equiv. PO43−/TEA salt, 8 equiv. ZnCl2, DMF, 3 h;
(v) 8 equiv. ZnCl2, DMF, 6 h.
Fig. 1 (A) Conversion of 2′-NH2-substituted cap analogue (1) into
2′-biotinylated cap analogue (2) via coupling with an in situ generated
NHS-biotin active ester. Reaction conditions: (i) O-(N-succinimidyl)-
1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), TEA, DMSO,
30 min; (ii) 0.5 M aqueous borane buffer pH 8.5, 1 h. (B) Structure of
cap analogues used in this work as reference compounds: standard cap
analogue, m7GpppG, and anti-reverse cap analogue, m27,2′-OGpppG.
HPLC, the reaction proceeded slowly. The conversion of the
starting material into the desired 5′-phosphorylated product
reached 95% after 8 h, whereas in the case of guanosine an
almost quantitative conversion under the same conditions
requires ca. 2 h. Compound 3 was isolated after ion-exchange
purification with an 80% yield. Subsequently, 3 was treated with
methyl iodide in DMSO to produce 2′-amino-2′-deoxy-7-
methylguanosine 5′-monophosphate (4). The methylation pro-
ceeded smoothly and occurred at the expected N7 position of
guanine, and not at the 2′-amino group of ribose, yielding 4 with
a 62% isolated yield. Compound 4 was then efficiently coupled
with GDP-imidazolide (6) in DMF in the presence of excess
ZnCl2 to produce cap analogue 1 (58% isolated yield).
The 2′-position of 7-methylguanosine in the dinucleotide cap
has been selected as a site for NH2 substitution and subsequent
biotin attachment, on the basis of the three factors listed above.
It has been well documented in the literature that 2′-O-methyl
substitution within the 7-methylguanosine of the dinucleotide
cap, similar to that at the 3′-O-methyl one, produces so-called
anti-reverse cap analogues (ARCA) that are incorporated into the
mRNA chain during transcription in vitro exclusively in the
correct orientation.22–24 In the X-ray structures of cap–eIF4E
complexes both 2′- and 3′-hydroxyls of 7-methylguanosine are
exposed to the solution and do not significantly contribute to
complex stabilization.25–27 Consequently, introduction of methyl
groups at either of these positions, as well as other, more bulky
substituents, was shown to have only a minor or moderate
influence on cap interaction with eIF4E and the translational
properties of capped-mRNA.22,23,28
Fig. 2 RP HPLC profiles from the reaction of 2′-NH2 substituted cap
analogue 1 with NHS-activated biotin at different time points: (A) 0 min
(before addition of NHS-biotin); (B) after 30 min (5 additions of 0.15
equiv. of NHS-biotin); (C) after 1 h (10 additions of 0.15 equiv. of
NHS-biotin). The peak designated with an asterisk (*) with Rt ∼ 3 min
is not a nucleotide derivative, but is associated with the presence of
DMSO and TSTU in the reaction mixture.
encompassed obtaining appropriate mononucleotide subunits 4
and 6, their subsequent coupling to the dinucleotide 1. To obtain
4, the starting material, 2′-amino-2′-deoxyguanosine, was first
phosphorylated with POCl3 in Yoshikawa conditions to yield the
corresponding 5′-monophosphate (3).21 As monitored by RP
Hence, we presumed that even the presence of a bulky biotin
residue at either the 2′- or 3′-position of cap 7-methylguanosine
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Table 1 Binding affinity of cap analogues 1 and 2 for eukaryotic
translation initiation factor 4E
Table 2 Translation efficiency and translational inhibition of mRNAs
encoding firefly luciferase and capped with cap analogues 1 and 2 in
rabbit reticulocyte lysate
Cap analogue
m7GpppG
KAS a [μM−1
]
Cap analogue at the
mRNA 5′ end
Relative translation
IC50 for m7GTPa
[μM]
9.4 0.4b
efficiencya
m27,2′-OGpppG
m7GNpppG (1)
m7GN-BiotpppG (2)
10.2 0.4b
5.6 0.6
3.8 0.1
m7GpppG
1
12.9 3.9
9.1 1.1
—
m27,2′-OGpppG
m7GNpppG (1)
m7GN-BiotpppG (2)
ApppG
1.4 0.2
0.9 0.1
1.0 0.1
0.16 0.03
a Determined in 50 mM Hepes–KOH (pH 7.2), 100 mM KCl, 0.5 mM
2.45 0.60
EDTA, 2 mM DTT at 20 0.2 °C. b Data from ref. 22.
n.d.b
a Determined in the rabbit reticulocyte lysate as described in ESI.† b The
value was not determined because m7GTP did not inhibit the translation
of this mRNA at the tested concentration range (1–100 μM).
would not completely disturb its interaction with eIF4E. When
comparing the 2′- and 3′-positions as potential sites of modifi-
cation, the former appeared to be more economical from the syn-
thetic point of view, since chemically synthesized 2′-modified
nucleosides are easier to prepare, and commercial ones less
expensive, than their 3′ isomers.
To evaluate whether selection of the 2′-position as a modifi-
cation site was correct, analogues 1 and 2 were tested as ligands
for the eIF4E protein by means of fluorescent quenching titration
experiments.27 The KAS values for cap–eIF4E were lower than
the value for corresponding unmodified analogues (Table 1).
Nonetheless, all the values were in the same order of magnitude,
which suggested that the analogues are bound by eIF4E in a
specific manner and, thus, may at least partially retain their bio-
logical functionality. In the case of analogue 2, the lower affinity
is likely a result of the presence of bulky biotin residue, whereas
in the case of analogue 1, it could rather result from an electro-
static repulsion between the protonated 2′-amino group and basic
amino acid side chains in the eIF4E–cap binding pocket.
The cap analogues 1 and 2 were then tested in the RNA poly-
merase catalyzed transcription reaction under conditions routi-
nely used for obtaining capped RNAs.24 First, we tested whether
the new cap analogues could be incorporated into short, 5-nt
transcripts by SP6 RNA polymerase. We found that 1 and 2 are
both incorporated into transcripts with efficiencies comparable to
m7GpppG and m27,2′-OGpppG and that the presence of the biotin
label could be indirectly observed by a slower migration com-
pared to other RNAs (ESI, Fig. S1†). Next, 1 and 2 were used as
reagents for the preparation of full-length mRNAs encoding
firefly luciferase. A typical reaction mixture contained dsDNA
template, ATP, CTP, UTP at 0.5 mM concentration, GTP at
0.1 mM concentration, SP6 RNA polymerase and either com-
pound 1 or 2 at 0.5 mM. Transcripts were then purified and ana-
lyzed by a non-denaturating 1% agarose gel electrophoresis
(ESI, Fig. S2†). The yields and homogeneities of all transcripts
were again comparable to those obtained by means of reference
cap analogues, m7GpppG and m27,2′-OGpppG.
The synthesized capped, full-length Luc-mRNAs were then
used to program the rabbit reticulocyte lysate system optimized
for cap-dependent translation to test their translational efficien-
cies. We have found that the Luc-mRNA capped with biotiny-
lated cap 2 (m7GN-BiotpppG-Luc-mRNA) is translated with an
efficiency lower than m27,2′-OGpppG-Luc-mRNA (Table 2). This
suggests that the biotin substituent, presumably due to its bulki-
ness compared to the 2′-O-methyl substituent, has reduced the
Nonetheless, m7GN-BiotpppG-Luc-mRNAs was translated with
an efficiency comparable to m7GpppG-Luc-mRNA, which is
more than 6-fold higher than the translation of mRNA bearing a
non-functional cap (ApppG) under these conditions. This
strongly suggests that m7GN-BiotpppG-Luc-mRNA is recruited
for the translational machinery by a cap dependent mechanism, i.
e. as in the majority of natural mRNAs. The translation
efficiency of mRNA capped with analogue 1, which bears a
non-bulky 2′-amino substituent, was also diminished compared
to m27,2′-OGpppG-Luc-mRNA (Table 2), which is in agreement
with the determined KAS value for the cap 1–eIF4E complex
(Table 1).
To gain more insight into how the biotin label influences the
functionality of the cap in the translation initiation process, we
tested if m7GN-BiotpppG-RNA is susceptible to inhibition by the
mononucleotide cap analogue, m7GTP. This compound is
known to be a potent inhibitor of cap-dependent translation,
acting by competitive blocking of mRNA cap binding to
eIF4E.27,29
Translation of all tested mRNAs capped with a functional
(m7G) cap was strongly inhibited by m7GTP (Table 2, ESI,
Fig. S3†). Interestingly, m7GN-BiotpppG-RNA had an IC50 value
3–5-fold lower than m7GpppG-RNA and m27,2′-OGpppG-RNA.
We believe that this confirms our hypothesis that cap 2 promotes
cap-dependent translation, but is less competitive for the trans-
lation machinery than e.g. m27,2′-OGpppG, and hence mRNAs
bearing this cap are more prone to inhibition by a free cap ana-
logue (m7GTP in this case).
Another set of experiments was conducted to confirm the
functionality of the biotin label introduced into RNA by capping
with analogue 2. An electrophoretic mobility shift assay in a
non-denaturating 1.4% agarose gel was used to follow the inter-
action of 140 nt m7GN-BiotpppG-RNA transcripts with recombi-
nant tetrameric streptavidin (Promega) (Fig. 4). In the presence
of streptavidin a fraction of the RNA pool migrated distinctly
slower than in its absence and the intensity of the slower-
migrating band was streptavidin concentration dependent.
Somehow unexpectedly, when a higher-resolution, automated
RNA separation system (Experion automated station, semi-dena-
turating conditions) was used to analyze the transcripts, we were
able to distinguish two different more slowly migrating bands,
whose relative ratio depended on the concentration of strepta-
vidin added to the RNA portion (ESI, Fig. S4†). We assume these
ability of cap
2 to compete for translation machinery.
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Conclusions
We developed a chemoenzymatic method for obtaining RNAs
that are specifically labelled with biotin at the 5′ m7G cap
moiety. The method encompasses co-transcriptional incorpor-
ation into the RNA transcript of a biotinylated, dinucleotide cap
analogue 2, obtained via chemical synthesis. The procedure is
applicable for labelling short RNA transcripts as well as full-
length mRNAs. We confirmed that mRNAs capped with the bio-
tinylated cap analogue 2 both undergo cap-dependent translation
and retain functionality of the biotin.
To introduce the biotin label at the 2′-amino group of the
cap’s 7-methylguanosine, we developed a simple and efficient
chemical procedure based on in situ generated NHS-biotin. The
high selectivity and efficiency of this reaction makes it a bene-
ficial option for introducing other tags bearing NHS-activatable
carboxyl groups, e.g. fluorescent ones.
The biotin labelled RNAs obtained by our method may be
applied to a variety of biological experiments based on biotin–
(strept)avidin technology or by means of biotin specific anti-
bodies, including protein affinity purification, pull-down assays,
in vivo visualization and many others. Particularly beneficial could
be their application for following eukaryotic mRNA cellular fate.
Finally, it is worth mentioning that cap analogue 1, bearing a
reactive 2′-amino group, once incorporated into transcripts
creates the possibility of post-transcriptional RNA labelling,
which is currently under investigation by our group.
Fig. 4 Electrophoretic mobility of transcripts capped in vitro with
m7GpppG or m7GpN-BiotppG (2) in the presence of streptavidin
(RNA-EMSA). Capped 140 nt RNA transcripts (2 pmol) were incubated
with increasing concentrations of tetrameric streptavidin (Promega).
Unbound RNA140 and formed RNA140–streptavidin complexes were
analyzed in 1.4% agarose gel in non-denaturating conditions as
described in Experimental procedures (for details see ESI† available
online): L1 – RNA size marker (from the bottom: 200, 500, 1000, 1500,
2000 nt). L2 – non-denaturated m7GpppG-RNA140 transcript, where
higher order bands of RNA140 are visible. L3 – thermally-denaturated
m7GpppG-RNA140. L4–L8 – binding reactions of m7GpppG-RNA140
with increasing concentrations of streptavidin. L9 – thermally-denatu-
rated m7GpN-BiotppG-RNA140
. L10–L13 – binding reactions of
m7GpN-BiotppG-RNA140 with increasing concentrations of streptavidin.
L14 – binding reaction of m7GpN-BiotppG-RNA140 with streptavidin in
the presence of competitive RNA (1750 nt luciferase mRNA). In all
cases, except L2, thermally-denaturated transcripts were used, which
migrate below the 200 nt size marker. Additionally, a less intense,
slower-migrating band corresponding to the refolded form of RNA140
Acknowledgements
We thank the Laboratory of Biological NMR (IBB PAS,
Warsaw) for access to the NMR apparatus and to Jacek Oledzki
from the Laboratory of Mass Spectrometry (IBB PAS) for
recording HRMS spectra. Financial support from the Polish
Ministry of Science and Higher Education (No. N204 089438,
No. N301 096339) is gratefully acknowledged.
was
observed
(compare
to
L2).
The
complexes
of
m7GpN-BiotppG-RNA140 with streptavidin (L11–L14) migrate slightly
slower than the refolded RNA. In L12–L14, the portion of unbound
RNA, observed even in the presence of high excess streptavidin, corres-
ponds to the non-capped (GTP initiated) RNA, whose presence is
typical for the synthesis of capped RNAs via co-transcriptional capping
(usually its content ranges from 10 to 20%).
Notes and references
‡A brief description of the activation procedure (for more experimental
details see the ESI†): To a solution of D-biotin (1.5 equiv., ∼0.25 M) in
DMSO were added TEA (2 equiv.) and TSTU (1.5 equiv.) and the
mixture was shaken at RT for 30 min. The resultant solution was added
portion-wise (10 additions over a period of 1 h) to a solution of
m7GNpppG (compound 1) (1 equiv., ∼0.1 M) in 0.5 M aqueous borane
buffer, pH 8.5. After each addition the pH was re-adjusted to 8.5 with
aqueous NaOH if necessary. The reaction progress was monitored by RP
HPLC. The reaction was quenched by dilution with water (∼4×) and
neutralization with a few drops of 50% acetic acid. The product was
purified by semi-preparative RP HPLC. Yield 60%.
bands correspond to RNA complexes with different oligomeric
forms of streptavidin, since the dissociation of streptavidin tetra-
mers in denaturating conditions is a known issue of the biotin–
streptavidin technology.30–32
We also tested whether the biotin label is recognized by strep-
tavidin in a more complex biological system, in which strepta-
vidin would be competing with other proteins, including those
capable of m7G cap binding. Different concentrations of strepta-
vidin were added to the rabbit reticulocyte lysate programmed
with either m27,2′-OGpppG-Luc-mRNA or m7GN-BiotpppG-Luc-
mRNA. The translation of m27,2′-OGpppG-mRNA remained
unaffected by streptavidin at all of the tested concentrations,
whereas translation of m7GN-BiotpppG-RNA was not inhibited by
streptavidin concentrations up to 8 nM, but was diminished 2–3-
fold in the presence of 20 nM tetrameric streptavidin, which cor-
responds to a ca. 1 : 2 RNA : streptavidin ratio (see ESI, Tables
S1 and S2 and Fig. S5 and S6†). We attribute this to the for-
mation of an m7GN-BiotpppG-RNA–streptavidin complex which
is formed in the RRL and impedes the translation initiation
process.
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