COMMUNICATION
Modified siRNAs for the study of the PAZ domainw
a
bc
bc
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Alvaro Somoza,* Montserrat Terrazas and Ramon Eritja*
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Received 16th February 2010, Accepted 5th May 2010
First published as an Advance Article on the web 19th May 2010
DOI: 10.1039/c003221b
Chemical modifications aimed at stabilizing the interaction
between the 30-end of siRNAs and the PAZ domain of RISC
have been tested for their effect on RNAi activity. Such
modifications contribute positively to the stability of siRNAs
in human serum.
that the presence of aromatic derivatives at the 30-end of the
guide strand could stabilize its interaction with the PAZ
pocket and therefore modulate the RNAi activity. Indeed,
it could be considered that stabilizing the complex between
the guide strand and the RISC complex would favour the
gene inhibition process. Under this hypothesis Ueno and
co-workers have explored the effect of aromatic modifications
at the 30-end of the guide strand on RNAi.9 However, no
significant differences were observed when siRNAs of the same
length (21 nucleotides) were compared. On the other hand,
crystal structures of the PAZ domain of Argonaute in complex
with DNA–RNA duplexes reveal that the 30-end is released
from the PAZ pocket during the cleavage of the mRNA.10 The
data suggest that this process is necessary to allow the protein
complex to adopt the conformation required to perform
RNase activity. Based on these results, one could expect
negative effects on RNAi activity for stable PAZ:guide-strand
complexes. In order to explore this hypothesis in more detail
we have modified the 30-end of the guide strand with aromatic
derivatives of diverse size, shape and electronic properties,
such as pyrene, anthracene, naphthalene, trifluoromethylbenzene
and fluorobenzene.
RNA interference (RNAi) is a powerful gene regulatory
process that allows the inhibition of genes in a very specific
way.1 This process is triggered by double stranded RNAs
known as small interfering RNAs (siRNAs)2 and is initiated
with the recognition of siRNAs by the RNA-induced Silencing
Complex (RISC), a protein complex located in the cytoplasm.
First, RISC binds the siRNA duplex,3 then, the passenger
strand is cleaved and released to the cytoplasm.4 On the other
hand, the complementary strand, known as the guide strand,
is used by the active complex to find the target sequence
along the messenger RNA (mRNA) by base complementarity
interaction. Finally, RISC induces endonucleotic cleavage
of the target mRNA, preventing its translation into the
corresponding protein.
During the last few years many efforts have been focused on
the use of this technology as targeted therapy, since it could be
useful for the treatment of diseases where the over-expression
of genes is involved, such as cancer or inflammation related
diseases.5 In order to develop an effective treatment it would
be desirable to achieve long lasting effects, good biodistribution
and high tissue, organ or cell selectivity.6 In this sense, several
groups have modified siRNAs with a broad spectrum of
chemical modifications in order to achieve long lasting effects
and to prevent the degradation of siRNAs by RNases.7
In this paper we study the effect of novel modifications at
the 30-end of the guide strand of siRNAs with the aim of
gathering structural information on the complex with RISC.
In particular, the binding to a hydrophobic pocket of the PAZ
domain of Argonaute protein (the key component of RISC)
involved in the recognition of the 30-end of the guide strand.
Crystallographic data reported for a fragment of the RISC
complex have shown the presence of aromatic residues at the
PAZ binding pocket.8 Based on these data one could expect
Moreover, the stability of the modified siRNAs in serum has
been evaluated.
The modified siRNAs at the 30-end were prepared using
functionalized Controlled Pore Glass (CPG) solid supports,
which were employed in the automated RNA synthesis.
Derivatives 1a–5a were prepared from L-threoninol and
the corresponding carboxylic acids (Scheme 1). Additionally,
acetic acid was utilized in the preparation of a non-aromatic
derivative (6a), which was used as control of the aromatic
interaction with the PAZ domain. The preparation of modified
siRNAs started with the addition of a carboxylic acid to
threoninol in the presence of the coupling reagents diisopropyl
carbodiimide and N-hydroxybenzotriazole in DMF, which
gave rise to the corresponding amide. The protection of the
primary hydroxyl function with the dimethoxytrityl group was
performed under standard conditions. Then, the solid supports
were functionalized with threoninol derivatives. The addition
of succinic anhydride and dimethylaminopyridine (DMAP)
to the threoninol derivatives yielded the corresponding
carboxylic acid. This group was activated by the addition of
2,20-dithiobis-(5-nitropyridine), DMAP, and triphenylphosphine
and utilized in the amide bond formation with the amino
groups present in the CPG support.11
a IMDEA-Nanociencia, 28049, Madrid, Spain.
E-mail: alvaro.somoza@imdea.org; Fax: +34 914976855;
Tel: +34 914973654
b
´
Instituto de Quımica Avanzada de Catalun˜a, CSIC,
C/Jordi Girona 18-26, E-08034, Barcelona, Spain
c
The modified solid supports were then employed in the
preparation of RNA strands using a RNA synthesizer.12
These strands were prepared to target the luciferase
gene, which encodes for a bioluminescent protein commonly
employed as reporter in gene inhibition assays.2 (Guide
`
Institut de Recerca Biomedica, CIBER-BBN, C/Baldiri Reixac 15,
E-08028, Barcelona, Spain. E-mail: ramon.eritja@irbbarcelona.org;
Fax: +34 932045904; Tel: +34 934039942
w Electronic supplementary information (ESI) available: Synthesis and
characterization of modified siRNAs. Complete gels. Experimental
details of RNAi evaluation. See DOI: 10.1039/c003221b
ꢀc
This journal is The Royal Society of Chemistry 2010
4270 | Chem. Commun., 2010, 46, 4270–4272
Somoza, Alvaro
