Click-modified anandamide siRNA enables delivery and gene silencing in neuronal and immune cells

Article abstract of DOI:10.1021/ja303251f

Click chemistry of alkyne-modified RNA with different receptor ligand azides was used to prepare 3′-folate, 3′-cholesterol, and, as a new entity, 3′-anandamide-modified RNA in high yields and excellent purity. The anandamide-modified RNA shows surprisingly high transfection properties and enables the delivery of siRNA even into difficult-to-transfect RBL-2H3 cells which model neuronal uptake. Furthermore, the system was employed in human immune cells (BJAB), demonstrating silencing effects similar to those of a cationic, benchmark transfection reagent. In addition, the anandamide conjugates were found to be nontoxic. The reported chemistry and the described properties of the anandamide siRNA extend the possibilities of using siRNA-based gene silencing in neuronal and immune cells.

Full text of DOI:10.1021/ja303251f

Communication
pubs.acs.org/JACS
Click-Modified Anandamide siRNA Enables Delivery and Gene
Silencing in Neuronal and Immune Cells
Julian Willibald,§,‡ Johannes Harder,§,‡ Konstantin Sparrer,∥ Karl-Klaus Conzelmann,∥
and Thomas Carell§
∥
§Center for Integrated Protein Science, Department of Chemistry, and Max von Pettenkofer-Institute and Gene Center,
Ludwig-Maximilians-Universitat, D-81377, Munich, Germany
̈
S
* Supporting Information
siRNA, despite the uncertainty that still exists regarding the
cannabinoid-mediated uptake mechanism.32,33 The synthesis of
the anandamide-modified RNA strand was performed as
depicted in Scheme 1. The central element of the synthesis is
the Cu-catalyzed alkyne−azide click reaction34−39 between an
alkyne-modified RNA strand and the corresponding ligand
azides 1. To compare the anandamide-modified RNA strands to
other systems, we utilized the click method also for the
preparations of a folate−RNA40 conjugate using folate azide 2
and of a cholesterol-modified RNA strand with the cholesterol
azide 3. We introduced in all cases a short tetraethyleneglycol
spacer between the RNA strand and the respective ligand. The
click technology enabled in all cases efficient ligation of the
hydrophobic and often quite insoluble (folate) ligand molecules
to RNA. In addition, the method enabled efficient conjugation
at the more difficult to access 3′-terminus of the siRNA duplex.
3′-Modified siRNA strands are typically better tolerated by the
RNAi machinery.41 To achieve the 3′-end attachment, we used
a deoxyuridine phosphoramidite with an octadiyne handle at
C5 during RNA synthesis.
The anandamide azide ligand 1 was prepared in just one step
from arachidonic acid 4 and the azido- and amino-function-
alized oligoethyleneglycol 5. The same strategy was employed
for the synthesis of the cholesterol azide 3. The folate derivative
2 was prepared via a slightly more elaborate synthesis starting
with the protected glutamic acid derivative 6, which was
condensed with the amino−azide tetraethyleneglycol com-
pound 5. Cleavage of the Fmoc group and coupling with
pteroic acid furnished, after deprotection, folate 2 (for more
details see SI). Compound 2 contains in this way the
ethyleneglycol spacer attached to the γ-carboxyl group, which
provides a folate compound with superior receptor binding
properties.42 The three azides were subsequently clicked with
excellent yields to the alkyne containing the RNA sense strand.
After HPLC purification, the ligand-modified RNAs were
hybridized to the antisense counterstrand to obtain the siRNA
duplexes depicted in Chart 1.
ABSTRACT: Click chemistry of alkyne-modified RNA
with different receptor ligand azides was used to prepare
3′-folate, 3′-cholesterol, and, as a new entity, 3′-
anandamide-modified RNA in high yields and excellent
purity. The anandamide-modified RNA shows surprisingly
high transfection properties and enables the delivery of
siRNA even into difficult-to-transfect RBL-2H3 cells which
model neuronal uptake. Furthermore, the system was
employed in human immune cells (BJAB), demonstrating
silencing effects similar to those of a cationic, benchmark
transfection reagent. In addition, the anandamide con-
jugates were found to be nontoxic. The reported chemistry
and the described properties of the anandamide siRNA
extend the possibilities of using siRNA-based gene
silencing in neuronal and immune cells.
NA interference (RNAi) is a powerful tool that utilizes
R21mer RNA double strands to repress the formation of a
particular protein in a cell.1−3 In nature, the silencing RNA
(siRNA) molecules are produced from larger transcripts that
are cut by the Dicer complex.4 For biotechnological application,
however, the siRNA molecules are chemically prepared and
administered. The idea to use siRNA as therapeutic agents5 has
been intensively pursued in the past decade, but the major
obstacle, poor cellular uptake of RNA duplexes, could not be
overcome.6 Currently, RNA delivery systems as divergent as
nanoparticles,7,8 liposomes,9,10 or polycation polymers11 are
under intensive investigation. Despite substantial progress in
the field, however, the often still high toxicity12−14 and low
cellular specificity represent problems that have yet to be
solved.15
Most recently, receptor-mediated endocytosis has evolved as
an alternative delivery strategy16−25 that allows targeting of the
siRNA to special cell types. The method requires linking the
siRNA to a ligand that binds to a cell-type-specific receptor.
This initiates an internalization process leading to the uptake of
the RNA−ligand conjugate. Currently, the strategy is most
successfully implemented with cholesterol-modified RNA.24
Here, we report that such a receptor-mediated strategy can be
successfully used to solve the problem that sensitive neuro-
nal26,27 and immune cells28 have been, up to now, difficult to
transfect. We discovered that the cannabinoid receptor present
on both cell types29 can be efficiently targeted with
arachidonoyl ethanol amide (anandamide)30,31-modified
To visualize the delivery of the RNA duplexes into live cells,
we initially hybridized the anandamide- and folate-modified
RNA sense strands to antisense strands containing a fluorescein
label. Figure 1 shows the result of confocal microscopy studies
performed with two different cells lines. For the anandamide-
modified RNA duplex we utilized RBL-2H3 cells, which serve
Received: April 4, 2012
Published: July 19, 2012
© 2012 American Chemical Society
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Communication
a
Scheme 1. Synthesis of Azide-Modified Anandamide 1 as Well as Folate (2) and Cholesterol (3) Derivatives
a
Reagents: 5 = 11-azido-3,6,9-trioxaundecan-1-amine, PyBOP = benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, TBTU = O-
(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate, DIPEA = N,N-diisopropylethylamine, DBU = 1,8-diazabicyclo[5.4.0]undec-7-
ene, 9 = pteroic acid, TFA = trifluoroacetic acid, CDI = carbonyldiimidazole.
Chart 1. Chemical Structure and Sequence of Anandamide- and Folate-Modified siRNA Targeting Renilla Luciferase
as a model for immune cell function.43 Barker et al. were able to
show that the uptake of anandamide by RBL-2H3 cells is
functionally identical to uptake by neuronal cells and
astrocytes.44 Thus this cell line is an excellent model for
anandamide uptake in immune cells and neurons.
Uptake of the folate-modified RNA duplex was studied with
HeLa cancer cells known to overexpress the folate receptor.
The microscopy studies showed that unmodified siRNA is, as
expected, unable to enter both cell lines. Anandamide- and
folate-modified siRNA, however, were readily detected inside
the respective cells, proving uptake. The same result was also
observed with modified dsDNA, which shows that the uptake is
entirely ligand dependent (Figure 1).
reporter assay. A plasmid containing two luciferases (Renilla
and Firefly) was transfected into the cells. RNAi was evaluated
by targeting the expression of the Renilla luciferase, whereas the
Firefly luciferase served as an internal standard. For these
studies we used the ligand-modified siRNA without transfection
reagent. Initial control experiments with unmodified RNA
duplexes (no ligand, no fluorescein) showed that the Renilla
expression was not affected. In contrast, we observed a dose-
dependent silencing of Renilla expression in presence of ligand-
modified siRNA in both cell lines (Figure 2a). Most
importantly, even a relatively low amount of ligand-modified
siRNA showed a considerable effect that ultimately led to a
relative silencing of about 60%. A final control experiment was
performed with a scrambled siRNA. Here again we were unable
to detect any luminescence reduction, showing that the
To demonstrate that the delivered siRNA exhibits the desired
RNAi effect, we utilized a commercially available dual-luciferase
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Journal of the American Chemical Society
Communication
system, which establishes the anandamide ligand as a powerful
new delivery tool.
To investigate if the anandamide−siRNA conjugate is able to
down-regulate a therapeutically important endogenous gene,
we next attempted to suppress the expression of the spleen
tyrosine kinase (SYK), which is a key protein involved in the
IgE-dependent inflammatory signaling cascades. As such, the
protein is a prospective target for the treatment of allergic and
inflammatory disorders.45,46 For the experiment we prepared an
ananamide-modified siRNA having the sequence described by
Sanderson et al.47 After addition of the siRNA conjugate to
RBL-2H3 cells, we monitored the expression level using real-
time PCR. Indeed, the expression of the SYK protein was
successfully reduced by about 55%, and again the anandamide
conjugate was found to be substantially more active than
cholesterol-modified siRNA (Figure 2).
To highlight the advantages associated with anandamide
conjugation, we compared the siRNA effect of anandamide-
modified siRNA with and without an additional transfection
agent (jetPRIME). The comparison was performed directly in
human immune B cells (BJAB) known to express the
cannabinoid receptors.48 The data depicted in Figure 3a show
Figure 1. Delivery of fluorescein-labeled siRNA and dsDNA to RBL-
2H3 and HeLa cells. Cannabinoid receptor expressing RBL-2H3 cells
were incubated with anandamide (AEA)-modified dsDNA and siRNA.
Folate receptor expressing HeLa cells were incubated with folate (FA)-
modified dsDNA and siRNA. As a negative control, both cell lines
were incubated with duplexes lacking a ligand modification.
Figure 3. (a) Relative silencing of Renilla luciferase mediated by
anandamide-modified siRNA and (b) relative toxicity of anandamide-
modified siRNA in the absence of jetPRIME (blue) and in the
presence of jetPRIME (gray) in human immune B cells (BJAB).
Quantification via luciferase luminescence.
that the silencing effect is in the same range, proving that
anandamide conjugation can replace the transfection agent. We
furthermore observed that anandamide conjugation increases
the activity of the jetPRIME-complexed siRNA by a factor of
almost 2 (Figure S3g). Most important, however, are the
toxicity data reported in Figure 3b, which show that the
anandamide conjugates are not toxic, in comparison to mock-
treated cells, which allows repeated treatment of the cells with
the conjugates (see also Figure S4).
Figure 2. (Top) Relative silencing of Renilla luciferase mediated by
(a) folate (FA)-modified siRNA in HeLa cells and (b) anandamide
(AEA)-modified siRNA in RBL-2H3 cells. Quantification via luciferase
activity. (Bottom) Relative silencing of (c) Renilla luciferase and (d)
spleen tyrosine kinase mediated by cholesterol (Chol, green)- and
anandamide (AEA, blue)-modified siRNA in RBL-2H3 cells.
Quantification via mRNA level.
In summary, we report here the use of the Cu-catalyzed
alkyne−azide chemistry for the construction of novel
anandamide−siRNA conjugates. The chemistry enables the
efficient construction of RNAs modified with different ligands
at the 3′-end in excellent yield and purity. This is particularly
noteworthy for folate-modified oligonucleotides, which are
notoriously difficult to access. The anandamide conjugation
allows transfection of immune cells and provides excellent
observed silencing is caused by specific binding of anandamide-
modified siRNA to the mRNA target. The silencing efficacy of
anandamide-modified siRNA was next evaluated in comparison
to that of the cholesterol−siRNA conjugate.24 The result of this
comparison is depicted in Figure 2. To our surprise, we noted
that the new anandamide 1-modified siRNA is constantly
significantly more potent than the broadly exploited cholesterol
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AUTHOR INFORMATION
Corresponding Author
Thomas.Carell@cup.uni-muenchen.de
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‡J.W. and J.H. contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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This work was supported by the Deutsche Forschungsgemein-
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