Diazirine-containing RNA photocrosslinking probes for the study of siRNA-protein interactions

Article abstract of DOI:10.1039/c0cc02450c

We here report the synthesis and characterization of small interfering RNAs with aryl trifluoromethyl diazirine moieties in the 3′-overhang regions, which allow sensitive detection of interacting proteins during assembly of the effector ribonucleoprotein complex by irradiation with minimally destructive long-wavelength ultraviolet light.

Full text of DOI:10.1039/c0cc02450c

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Diazirine-containing RNA photocrosslinking probes for the study
of siRNA–protein interactionsw
a
cd
Satoru Kuboe,z Mayuko Yoda,z Aya Ogata,a Yukio Kitade,ab Yukihide Tomari*cd and
Yoshihito Ueno*ab
Received 9th July 2010, Accepted 16th August 2010
DOI: 10.1039/c0cc02450c
We here report the synthesis and characterization of small
interfering RNAs with aryl trifluoromethyl diazirine moieties
in the 30-overhang regions, which allow sensitive detection of
interacting proteins during assembly of the effector ribonucleo-
protein complex by irradiation with minimally destructive long-
wavelength ultraviolet light.
Fig. 1 Structures of functional compounds used in this study.
RNA interference (RNAi) is a process of sequence-specific
posttranscriptional gene silencing that is triggered by double-
stranded RNAs (dsRNAs).1 This pathway is initiated by
processing of dsRNA into 21- to 23-nucleotide (nt) duplexes
by Dicer. These small RNA duplexes, which contain a 2-nt
overhang at the 30-end of each strand, are termed small
interfering RNAs (siRNAs). siRNAs are incorporated into
the effector ribonucleoprotein complex, termed RNA-induced
silencing complex (RISC), which catalyzes the sequence-specific
cleavage of target mRNAs.1,2
The cleaved passenger strand is discarded and rapidly
degraded, with only one strand (the guide strand) remaining
in Ago2. This process is termed unwinding, and the Ago2
protein containing only the guide strand is called mature
RISC, or simply, RISC. Although much is known about RISC
assembly of fly Ago2, the protein factors that are required for
this entire pathway are not fully understood.3
Photocrosslinking has been a tremendously useful technique
to study RISC assembly. For example, asymmetric binding of
Dcr-2/R2D2 in RLC to the siRNA duplex, and transition
from Dcr-2/R2D2 in RLC to Ago2 in RISC can be monitored
by using the photocrosslinker 5-iodouridine (3) introduced at
position 20 of the guide strand (Fig. 1).7 However, the
A member of the Argonaute (Ago) family of proteins lies at
the heart of RISC, which provides the unique platform for
target recognition and cleavage. RISC assembly is not a mere
binding of siRNAs to Ago proteins, but rather follows a
complex, multi-step pathway.3 In Drosophila melanogaster, it
is well established that siRNA duplexes are first incorporated
into RISC-loading complex (RLC), which contains Dicer-2
(Dcr-2) and R2D2.4–6 RLC senses the thermodynamic
asymmetry of the siRNA duplex, with R2D2 located at the
more stable end and Dcr-2 at the less stable end, and ascertains
that the strand with the less stable 50 end is eventually selected
as the guide in mature RISC.7 Ago2 then receives the siRNA
duplex with the predetermined orientation from RLC. This
process is called RISC loading and requires ATP and the
Hsc70/Hsp90 chaperone machinery.8 After RISC loading,
Ago2 cleaves the center of one strand (the passenger strand)
of the siRNA duplex, just like it cleaves target mRNAs.9–11
a Department of Biomolecular Science, Faculty of Engineering,
Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
E-mail: uenoy@gifu-u.ac.jp; Fax: +81-58-293-2794;
Tel: +81-58-293-2639
b United Graduate School of Drug Discovery and Medical Information
Sciences, Gifu University, Japan
c Institute of Molecular and Cellular Biosciences, The University of
Tokyo, Tokyo, 113-0032, Japan. E-mail: tomari@iam.u-tokyo.ac.jp;
Fax: +81-3-5841-8485; Tel: +81-3-5841-7839
Scheme 1 Reagents and conditions: (a) CF3CO2Et, n-BuLi, THF, ꢀ78 1C,
1 h, 67%; (b) (1) HONH2ꢁHCl, EtOH/pyridine (1 : 1, v/v), 60 1C, 12 h;
(2) TsCl, Et3N, DMAP, CHCl3, rt, 12 h, 85%; (c) NH3, THF, rt, 48 h,
60%; (d) I2, Et3N, MeOH, rt, 30 min, 74%; (e) TBAF, THF, rt,
2 h, 85%; (f) DMTrCl, pyridine, rt, 5 h, 38%; (g) (1) succinic
anhydride, DMAP, pyridine, rt, 24 h; (2) CPG, EDCI, DMF, rt,
48 h, 28 mmol gꢀ1 (loading amount).
d Department of Medical Genome Sciences, The University of Tokyo,
Japan
w Electronic supplementary information (ESI) available: General
experimental section, protocols for synthesis, purification and
characterization of aromatic compounds and ONs, and protocols
for biological experiments. See DOI: 10.1039/c0cc02450c
z These authors contributed equally to this work.
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 7367–7369 7367

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crosslinking efficiency of 5-iodouridine is limited, and irradiation
with ultraviolet (UV)-B (280–315 nm) to activate 5-iodouri-
dine poses a risk of damaging biomacromolecules and their
interactions. Therefore, an alternative photocrosslinker with
higher crosslinking efficiency at a longer wavelength has long
been awaited.
Aromatic azides, aromatic diazirines, and benzophenones
are representative photoreactive functional groups that
generate highly reactive species such as nitrenes, carbenes,
and diradicals, respectively, by irradiation with light. Among
them, aromatic azides are the most widely used because of
their synthetic simplicity.12 On the other hand, aromatic
diazirines are considered the most effective functional group
because they generate carbenes, which can react with various
chemical groups including the generally inactive C–H bond.13
Aromatic diazirines are also preferred because they are photo-
activatable with long-wavelength UV-A (>315 nm), which is
less likely to damage biomacromolecules than UV-B. It has
been shown that diazirine–DNA conjugates are useful for the
study of DNA–protein interactions.14
We have recently reported the synthesis of siRNA possessing
an aromatic compound 1,3-bis(hydroxymethyl)benzene (1) in
the 30-overhang region (Fig. 1).15 We found that this modified
siRNA was more potent in terms of target gene silencing than
the siRNA without 30-overhang regions; further, the modified
siRNA had silencing activity equivalent to that of siRNA
possessing natural nucleosides at the 30-overhang region. On
the basis of this information, we report the synthesis and
characterization of siRNA with aryl trifluoromethyldiazirine
moieties 2 in the 30-overhang region (Fig. 1); these moieties
show robust photocrosslinking to siRNA-interacting proteins on
irradiation with minimally destructive long-wavelength UV.
1,3-Bis(tert-butyldimethylsilyloxymethyl)-5-iodobenzene
(4), which was prepared as described previously,16 was
trifluoroacetylated by lithium halogen exchange at ꢀ78 1C
using n-BuLi followed by addition of ethyl trifluoroacetate
Fig. 2 Sequences of the oligonucleotides (ONs) and siRNA used in
this study. Capital letters indicate ribonucleosides, whereas the small
italicized letters show 20-deoxyribonucleosides. B denotes 1,3-bis-
(hydroxymethyl)benzene. Z and X indicate aryl diazirine and 5-iodouridine,
respectively. In the case of siRNAs 4–7, the upper strands were
50 radiolabeled with 32P (highlighted in yellow), whereas the lower
strands bore nonradioactive 50 phosphate.
respectively. These ONs were analyzed by matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF/MS), and the observed molecular weights were
in agreement with their structures.
We first examined the silencing activity of siRNA carrying
the aryl diazirine in its 30-overhang region by a dual-reporter
assay using a psiCHECK-2 vector in HeLa cells. The vector
contains the Renilla and firefly luciferase genes, and the siRNA
sequences were designed to target the Renilla luciferase gene.
HeLa cells were co-transfected with the vector and indicated
amounts of the siRNA, and the signals of Renilla luciferase
were normalized to those of firefly luciferase. The silencing
activity of siRNA with the aryl diazirine (siRNA 3) was found
to be comparable to that of siRNA possessing the natural
nucleoside (siRNA 1) or 1 (siRNA 2) in the 30-overhang region
(Fig. 3).
(Scheme 1). The resulting trifluoroacetophenone
5 was
converted to a stereoisomeric (E/Z) mixture of oximes by
treatment with HONH2ꢁHCl. The crude mixture of oximes
was tosylated to generate a mixture of tosyl-oximes 6. This
mixture was converted to diaziridine 7 upon addition of
ammonia, and 7 was oxidized to the corresponding diazirine
by using I2. Deprotection of the silyl ether using tetra-n-
butylammonium fluoride (TBAF) afforded the benzylic
alcohol 9. One out of the two hydroxy groups of 9 was
protected by a 4,40-dimethoxytrityl (DMTr) group to produce
a mono-DMTr derivative 10. To enable attachment to the
solid support, 10 was succinated to yield the corresponding
succinate, which was linked to controlled-pore glass (CPG) to
afford the solid support 11 possessing 10. siRNA sequences
used in this study are depicted in Fig. 2.
All the oligonucleotides (ONs) were synthesized by a phospho-
ramidite method. Fully protected ONs (1.0 mmol each) were
treated with concentrated NH4OH : EtOH (3 : 1, v/v) at room
temperature for 12 h and then with 1.0 M TBAF/THF at room
temperature for 12 h. The ONs released after the treatment
were purified by denaturing 20% polyacrylamide gel electro-
phoresis (PAGE) to afford deprotected ONs 5 and 9 carrying
the aryl diazirine in 14 and 11 OD260 absorbance units,
Fig. 3 Dual-luciferase assay.
c
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Fig. 4 Superior photocrosslinking efficiency of aryl diazirine compared to 5-iodouridine. (a) Photocrosslinking of the siRNAs to RISC assembly
factors with 302 nm UV-B irradiation. (b) Photocrosslinking of the siRNAs to RISC assembly factors with 365 nm UV-A irradiation.
siRNAs 4 and 5 contain the conventional 5-iodouridine at
position 20 in the 30-overhang region of the upper strand as a
control, whereas siRNAs 6 and 7 contain the aryl diazirine 2 at
position 21 of the upper strand (shown in red) (Fig. 2).
siRNAs 4 and 6 have a mismatch at the 50 end of their
respective upper strands; therefore, the crosslinker-containing
strand serves as the guide in mature RISC. In contrast,
siRNAs 5 and 7 have a mismatch at the 50 end of their
respective lower strands (shown in blue); therefore, the
crosslinker-containing strand is discarded during unwinding.
In the case of all the siRNAs, we radiolabeled the 50 ends
This work was in part supported by a grant from Precursory
Research for Embryonic Science and Technology (PRESTO)
of the Japan Science and Technology Agency.
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a
Drosophila embryo lysate for the indicated time, irradiated the
lysate with 302 nm UV-B or 365 nm UV-A, and separated the
crosslinked proteins by sodium dodecyl sulfate (SDS)-PAGE.
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c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 7367–7369 7369