Chimeric RNA Oligonucleotides with Triazole and Phosphate Linkages: Synthesis and RNA Interference

Article abstract of DOI:10.1002/asia.201500765

Chimeric RNA oligonucleotides with an artificial triazole linker were synthesized using solution-phase click chemistry and solid-phase automated synthesis. Scalable synthesis methods for jointing units for the chimeric structure have been developed, and after click-coupling of the jointing units with triazole linkers, a series of chimeric oligonucleotides was prepared by utilizing the well-established phosphoramidite method for the elongation. The series of chimeric 21-mer oligonucleotides that possessed the triazole linker at different strands and positions allowed for a screening study of the RNA interference to clarify the preference of the triazole modifications in small-interfering RNA molecules.

Full text of DOI:10.1002/asia.201500765

DOI: 10.1002/asia.201500765
Full Paper
RNA Modification
Chimeric RNA Oligonucleotides with Triazole and Phosphate
Linkages: Synthesis and RNA Interference
Tomoko Fujino,[a] Kanako Kogashi,[a] Koudai Okada,[a] Martin Mattarella,[a] Takeru Suzuki,[a]
Kenichi Yasumoto,[b] Kazuhiro Sogawa,[b] and Hiroyuki Isobe*[a, c]
Abstract: Chimeric RNA oligonucleotides with an artificial tri-
azole linker were synthesized using solution-phase click
chemistry and solid-phase automated synthesis. Scalable
synthesis methods for jointing units for the chimeric struc-
ture have been developed, and after click-coupling of the
jointing units with triazole linkers, a series of chimeric oligo-
nucleotides was prepared by utilizing the well-established
phosphoramidite method for the elongation. The series of
chimeric 21-mer oligonucleotides that possessed the triazole
linker at different strands and positions allowed for a screen-
ing study of the RNA interference to clarify the preference of
the triazole modifications in small-interfering RNA molecules.
Introduction
Analogues of oligonucleotides are both attractive synthetic tar-
gets for chemists and interesting tools for biologists. Phos-
phate-replaced analogues that possess furanose-mimicking
rings are particularly interesting because they can serve as sub-
strates for enzymatic reactions despite their inertness toward
nuclease hydrolysis.[1,2] Because of the robust elongation reac-
tions of the so-called click chemistry,[3] triazole-linked DNA ana-
logues (TLDNA) have fulfilled the criterion for oligomer synthe-
sis[2,4] and have proven useful in several enzymatic reactions.[5,6]
Recently, we introduced an RNA analogue with triazole linkag-
es (TLRNA) by designing an elongation unit that possesses 3’-
azido and 5’-acetylene moieties (Figure 1).[7,8] The synthetic
access to this elongation unit was far superior to that of TLDNA
because of the concise synthesis route from d-xylose as well as
the selective and easy process for the nucleobase introduction,
which allowed for the synthesis of oligomers.[2,9] However, ex-
ploration of the biological functions of the TLRNA oligomers
Figure 1. Structures of the triazole-linked RNA for the chimeric oligonucleo-
tides.
was severely hampered by the poor solubility of the fully
modified, electroneutral oligonucleotides. Therefore, we decid-
ed to incorporate the triazole linkages in the form of chimeric
RNA oligonucleotides with a combination of natural, charged
phosphate linkages. Scalable synthesis methods for jointing
units for the chimeric structure have been developed, and
after click-coupling of the jointing units with triazole linkers,
a series of chimeric oligonucleotides was prepared by utilizing
the well-established phosphoramidite method for the elonga-
tion.[10] The series of chimeric oligonucleotides that possess the
triazole linkage at different positions provided us with a screen-
ing study of the RNA interference (RNAi) to clarify the tolerance
of the triazole modifications in small interfering RNA
(siRNA).[11,12]
[a] Dr. T. Fujino, K. Kogashi, K. Okada, Dr. M. Mattarella, T. Suzuki,
Prof. H. Isobe
Department of Chemistry
Tohoku University
Aoba-ku, Sendai, 980-8578 (Japan)
E-mail: isobe@m.tohoku.ac.jp
[b] Dr. K. Yasumoto, Prof. K. Sogawa
Department of Biomolecular Sciences
Tohoku University
Aoba-ku, Sendai, 980-8578 (Japan)
[c] Prof. H. Isobe
JST, ERATO, Isobe Degenerate p-Integration Project and Advanced Institute
for Materials Research
Tohoku University
Aoba-ku, Sendai, 980-8577 (Japan)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/asia.201500765.
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Results and Discussion
Similar to the elongation units for the TLRNA,[7] synthesis routes
from the readily available d-xylose were developed for the 5’-
and 3’-jointing units. Because the synthesis routes intrinsically
follow the preceding transformation processes, we briefly de-
scribe the design principles for each unit and provide the
entire synthesis methodology in the Supporting Information.
The 5’-jointing unit requires protecting groups on the nucleo-
bases and on the 2’-OH and 5’-OH moieties, and it requires the
3’-azido group. Furthermore, the automated protocols for the
phosphoramidite elongation required the presence of the 4,4’-
dimethoxytrityl (DMTr) group on the 5’-OH moiety (see below).
Thus, a series of compounds 1 that possessed four different
nucleobases (U, C, A and G) was synthesized from d-xylose in
high yields (Figure 2; see also Scheme S1 in the Supporting In-
Scheme 1. Synthesis of the triazole-linked dinucleotides. The triazole linkage
is represented as t.
cation of target sites in the EYPF gene are shown in Figure S52.
For siRNA-a, oligonucleotide 7 possessed a sequence for a pas-
senger strand, and oligonucleotide 8 possessed a sequence for
a guide strand. For siRNA-b, oligonucleotide 9 was the passen-
ger strand, and oligonucleotide 10 was the guide strand.
The triazole-linked dinucleotide was incorporated using the
phosphoramidite method that was executed on an automated
synthesizer (M-2-MX, Nihon Techno Service) with controlled
pore glass (CPG) beads, and the representative steps for the in-
corporation are shown in Scheme 2. For the 3’-jointing process
for the initial chimeric structure 4, the phosphoramidite end of
the dinucleotide was activated with 5-benzylthio-1H-tetrazole
(BTT) to couple with the hydroxy end either of a nucleoside or
of a universal linker[15] on the CPG beads. The subsequent pro-
cesses were identical to those for the standard phosphorami-
dite protocol[10] and involved capping of the unreacted hy-
droxy ends, oxidation of the phosphorous moieties and depro-
tection of the 5’-end DMTr group. For the 5’-jointing process
to elongate the natural nucleotides, the standard phosphora-
midite method was adopted with the natural nucleoside 5,
which was repeated to complete the desired sequence. The ef-
ficiency of elongation was tracked for each step with an LED
detector implemented on the synthesizer and was finally de-
termined at the final detritylation process using the standard
protocol to quantify the trityl cation on a UV/Vis spectrometer.
The synthesis of the 21-mer involved 19/20 elongation steps
and, as shown in Scheme 2, the presence of the triazole-linkag-
es did not affect the efficiency: the total yields of the oligonu-
cleotides ranged from 30% to 60%, which required very high
efficiencies of 94–97% per one elongation step.
Figure 2. The jointing units synthesized in this study.
formation). The 3’-jointing unit requires protecting groups on
the nucleobases, on the 2’-OH moiety, and on the acetylene
unit at the 5’-position, and it requires a free 3’-OH moiety. Be-
cause of a potential cyclization reaction that can bridge the 2’-
and 3’-OH groups, the protecting group for the 2’-OH was
requisitely a silyl protecting group.[13] Therefore, compound 2
was designed and synthesized with four different nucleobases
(U, C, A and G) in high yields (Figure 2; see also Scheme S2).
The two jointing units were coupled to produce the simplest
triazole-linked dinucleotides for examination in the automated
phosphoramidite protocols (n=0, Figure 1). Among the 16
possible structures for the dinucleotides, we selected three
representative examples from a combination of pyrimidines for
this first study. As shown in Scheme 1, we coupled 1 and 2 by
a copper-catalyzed Hüisgen [3+2] cycloaddition reaction and
subsequently converted the dinucleotides into phosphorami-
dites. The yields were moderate to good for the two-step
transformation, and three dinucleotides, UtU, CtU and CtC,
were synthesized in 87%, 77% and 80% yields, respectively.
We incorporated the triazole-linked dinucleotide in a 21-mer
oligonucleotide with an siRNA sequence to target a gene of
enhanced yellow-fluorescent protein (EYFP).[14] Using dimers in
our hands, we designed two siRNA, siRNA-a and siRNA-
b (Scheme 2), for the EYFP target gene and synthesized 13 oli-
gonucleotides varying the positions of modification.[11] The lo-
Finally, the activity of the chimeric oligonucleotides as siRNA
was examined. We assembled the double-stranded siRNA by
combining the passenger strand (7 or 9) and the guide strand
(8 or 10)[16] and introduced it into HeLa cells. A plasmid pEYFP-
N1 with the target EYFP gene was also transfected with a plas-
mid pTagRFP-C that carried a reference gene of red fluorescent
protein (tagRFP) as the internal standard. The reduction in the
target EYFP expression was quantified by quantitative RT-PCR
of the corresponding mRNA in comparison with the standard
tagRFP expression (see the Experimental Section for the de-
tailed procedures).[5,17]
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modification at 3–4 counting
from the 5’-end interfered the
target gene at 7% expression
level. This interference level was
comparable with that of the
siRNA-b with natural oligonu-
cleotide 9-nat of 4% expression
level. As was the case with RNA-
a, the triazole linkers were favor-
ably tolerated in the down-
stream region, and modifications
in the 3’-overhang regions at
19–20 and 20–21 counting from
the 5’-end with 9-iii and 9-iv
suppressed the expression levels
to 10% and 6%, respectively. Be-
cause the interference with the
7-i/8-vii
combination
(Fig-
ure S53) was not more potent
than those of 9-i/10-nat or 9-
nat/10-iv combinations, the chi-
mera/chimera combination of
two modified oligonucleotides
with a triazole linker may not be
effective for a synergetic im-
provement of interference.[12d–f]
However, the modifications in
the upstream region considera-
bly affected the siRNA activity.
The oligonucleotide 10-i exhibit-
Scheme 2. Synthesis of 21-mer oligonucleotides for the RNAi assay.
ed negligible interference activi-
ty. The triazole modification in
this oligonucleotide was located
The sequences of siRNA-a and siRNA-b were selected so that
we could cover a variety of modification sites with our pyrimi-
dine dimers. Note that modifications in the guide strand of the
siRNA are regarded as more challenging,[12] and we examined
a larger number of variants in the guide strand. The locations
of the triazole modifications in terms of counting from the 5’-
end in siRNA were 2–3, 3–4, 12–13, 14–15, 15–16, 17–18 and
20–21 for the guide strand 8 in siRNA-a and 5–6, 8–9, 19–20
and 20–21 for the guide strand 10 in siRNA-b. The oligonucle-
otides were screened for the siRNA activity via two stages,
and, at the first stage, a rough screening of modification sites
was executed by a single-set experiment for 13 oligonucleo-
tides. As shown in Figure S53, most of the modifications in
RNA-a were not tolerated except the 3’-overhang region at the
20–21 counting with 7-i/8-vii combination. Note that the ex-
amination of the 3’-overhang region was also included in RNA-
b and that the in-depth evaluation was performed with RNA-
b (see below). At the second stage, we further accumulated
data for potent candidates (RNA-b) in the first stage with ex-
periments being executed in triplicate to obtain reliable and
quantitative data for the positive experiments (Figure 3).
between nucleotides of 5–6 counting from the 5’-end. This
region of 2–8 counting from the 5’-end is known as the seed
region where the hydrogen bonding from the Argonaute pro-
tein captures the phosphate groups[18] and the complementari-
ty of base pairing with the target strand plays a crucial role for
the siRNA activity.[12] The oligonucleotide 10-ii with the triazole
modification between nucleotides of 8–9 counting from the 5’-
end indeed exhibited a moderate level of interference and
suppressed the expression to 46%. A dose dependency of the
siRNA activities was evaluated with the most potent chimeric
siRNA of 9-nat/10-iv. As shown in Figure 3b, the influence of
the concentration on the interference was more apparent with
9-nat/10-iv than the natural combination of 9-nat/10-nat.
Finally, Figure 4 shows the results of analysis of the translat-
ed products by fluorescent microscopy. A comparison of
tagRFP with 4’,6-diamidino-2-phenylindole (DAPI) shows the
transfection efficiency of the plasmids with marker genes, and
a comparison of EYFP with tagRFP shows the RNAi activity. A
further comparison of EYFP in control with those in 9-nat/10-
nat or 9-nat/10-iv clarifies the RNAi activities of the chimeric
siRNA. The chimeric siRNA 9-nat/10-iv indeed suppressed the
production of the target EYFP at the level comparable to the
natural congener 9-nat/10-nat, which is consistent with the re-
sults from quantitative PCR analysis. The results confirmed that
As is typical with artificial oligonucleotides,[12d–f] the modifica-
tion in the passenger strand was well tolerated to achieve
siRNA activity, and the passenger strand 9-i with a triazole
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the chimeric RNA oligonucleotide possessing a triazole linkage
functioned as siRNA and, albeit depending on the installed po-
sitions, that the silencing activities could compete with the
natural RNA oligonucleotides.
Conclusions
In summary, we have developed a concise synthesis method
for nucleoside analogues that serves as jointing units for chi-
meric RNA oligonucleotides with triazole linkages. In total, 48
compounds including 13 chimeric oligonucleotides were newly
synthesized. In combination with four elongation units,[7] eight
jointing units that possess four different nucleobases can pro-
vide a synthesis route to any sequence of oligonucleotides
with triazole linkages at any desired position. Note that the
method allows for the introduction of non-natural nucleobases
through glycosidation reaction with the corresponding nucleo-
base units. The present study also demonstrates the applicabil-
ity of the triazole-linked nucleotides for the automated proto-
cols of phosphoramidite methods, which allow for the incorpo-
ration of the triazole linker in 21-mer oligonucleotides for
siRNA. The chimeric oligonucleotides were examined for the
RNAi activity, and we revealed the preference of the triazole
modifications, which added a new, synthetically viable entry
into the rapidly growing library of siRNA variants.[12] We believe
that the present synthesis method will lead to the develop-
ment of various functional RNA molecules in the future.
Figure 3. Performance of the siRNA as measured by EYFP gene expression in
HeLa cells in comparison with the RFP gene as the internal standard. Aver-
age data with error bars from triplicate runs are shown. (a) Dependency of
siRNA activities on the triazole modification positions at the concentration
of 10 nm of siRNA. (b) Dependency of siRNA activities on the concentrations
as measured with the most potent siRNA-b (9-nat/10-iv). The total concen-
tration of transfected RNAs was maintained at 10 nm, and the additional
non-targeting RNA was introduced for the experiments with siRNA below
10 nm.
Experimental Section
General
All the reactions were carried out under an inert atmosphere of ni-
trogen unless otherwise noted. Analytical thin-layer chromatogra-
phy (TLC) was performed on a glass plate coated with silica gel
(230–400 mesh, 0.25 mm thickness) containing a fluorescent indica-
tor (silica gel 60F254, Merck). Flash silica gel column chromatogra-
phy was performed on 60N (spherical and neutral gel, 40–50 mm,
Kanto).[19] Elongation of chimeric oligonucleotides was performed
on a Nihon Techno Service M-2-MX DNA/RNA synthesizer. Analysis
of reaction mixtures with high pressure liquid chromatography
(HPLC) were performed on HPLC systems equipped with ODS
column (COSMOSIL C18-MS-II, 4.6”250 mm, Nacalai Tesque;
column temperature 408C), and purification of products was per-
formed with ODS column (COSMOSIL C18-MS-II, 20”250 mm, Na-
calai Tesque). IR spectra were recorded on NICOLET iS10 equipped
with an attenuated total reflection (ATR; powder) and were report-
ed as wavenumbers (n) in cmÀ1. Proton (1H) and carbon (13C) nucle-
ar magnetic resonance (NMR) spectra were recorded on JEOL JNM-
ECS 400 (1H: 400 MHz; 13C: 100 MHz; 31P: 160 MHz) spectrometers.
Methyl (CH3), methylene (CH2) and methyne (CH) signals in
13C NMR spectra were assigned by DEPT spectra. 1H NMR spectra
and 13C NMR spectra in CDCl3 were referenced to the solvent reso-
nances, and 31P NMR spectra were referenced to 85% H3PO4 as an
external standard. High resolution mass spectra were obtained on
JEOL JMS-T100 LC or SolariX 9.4T instrument (ESI-TOF MS) with re-
serpine (1 ngmLÀ1) and a mixture of polyethylene glycol (PEG200
20 ng, PEG600 20 ng, PEG1000 30 ng, PEG2000 60 ng in 1 mL) as an
internal standard. A molar amount of trityl cation at the final elon-
gation step was quantified on a UV-visible spectrometer (JASCO, V-
Figure 4. Fluorescent microscopy images for the translated products of EYPF
(green) and tagRFP (red). Two siRNA, 9-nat/10-nat and 9-nat/10-iv, target
the EYFP gene. Images (blue) with DAPI staining for non-selective, nuclear
staining are also shown. A control experiment was carried out with non-tar-
geting RNA, and a reference experiment was carried out with natural siRNA
9-nat/10-nat. The experiment with chimeric siRNA 9-nat/10-iv showed
a comparable suppression of EYFP production with the natural reference via
RNAi. The images shown are representative of three independent experi-
ments. Scale bar, 100 mm.
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670) to determine the elongation efficiencies. Thermal melting
curve was also obtained on the spectrometer equipped with
a water-circulated temperature-controlled cell holder (JASCO, ETC-
717). The melting temperature was determined using a melting
program (JASCO, VWTP-780). Quantitative PCR was performed on
ABI 7300 Real Time PCR System (Applied Biosystems), and the
levels of PCR products were analyzed with 7300 System SDS soft-
ware (Applied Biosystems). Fluorescent microscopic analysis was
performed by using an Olympus IX-71 system equipped with color
filters of U-MYFPHQ (EYFP), U-MRFPHQ (tagRFP) and U-MWU2
(DAPI) on an Olympus DP70 CCD camera.
908C for 1 min and allowed to cool to 308C over a period of
25 min. The structures of siRNA were shown in main text. For a neg-
ative control, non-targeting RNA duplex with 5’-CpGpApApUpCpC-
pUpApCpApApApGpCpGpCpGpCpdTpdT-3’
and
5’-
GpCpGpCpGpCpUpUpUpGpUpApGpGpApUpUpCpGpdTpdT-3’ was
prepared.
RNAi experiments
HeLa cells were cultured in E-MEM supplemented with 10% v/v
FBS and 1% v/v penicillin-streptomycin at 378C with 5% CO2,[11]
and the growing cells in E-MEM with 10% v/v FBS (1.5”
105 cellsmLÀ1) were plated into each well of 12-well plates (1 mL/
well) and incubated for 24 h prior to transfection. The cells were
then transfected with a mixture of pEYFP-N1 plasmid (0.15 mg),
pTagRFP-C plasmid (0.15 mg) and siRNA duplex (10 pmol) using lip-
ofectamine 2000 (2.0 mL). After 4 h, the medium was exchanged
with fresh growth medium containing 10% v/v FBS. The cells were
allowed to grow for 48 h and the gene expression levels were ana-
lyzed by quantitative RT-PCR and fluorescent microscopy in terms
of mRNA transcripts and translated proteins, respectively.
Materials
Anhydrous THF (stabilizer free, Kanto), DMF (dehydrated, Kanto)
and toluene (dehydrated, Kanto) were purified by using a solvent
purification system (GlassContour) equipped with columns of acti-
vated and supported copper catalyst (Q-5). Water was purified by
using a Milli-Q ultrapure water system (Millipore). Other solvents
were purified by distillation and dried over 4 Š molecular sieves. d-
Xylose, triflic acid, sodium azide, N,N-dimethyl-4-aminopyridine
(DMAP), pyridinium dichromate (PDC), (triisopropylsilyl)acetylene,
uracil, N,O-bis(trimethylsilyl)acetamide, 9-acetyl-2-(acetylamino)-6,9-
dihydro-1H-purin-6-one, triethylamine, Eagle’s minimal essential
medium (E-MEM) and penicillin-streptomycin solution were pur-
chased from Wako, and triethylamine was purified by distillation
before use. Benzyl bromide, sodium borohydride and tert-butyllithi-
um were obtained from Kanto, and tert-butyllithium was titrated
before use. Triflic anhydride, hexamethyldisilazane, trimethylsilyl tri-
flate, boron trichloride, n-tetrabutylammonium fluoride, tert-butyl-
dimethylsilyl chloride, imidazole and cytosine were purchased from
TCI. n-Tetrabutylammonium iodide, adenine, benzoyl cytosine, pri-
mers, DAPI and fluorescein amidite (FAM)-labeled Taqman probes
used for quantitative PCR were purchased from Aldrich. Diisopro-
pylethylamine, trimethylsilyl chloride, dimethoxytrityl chloride,
boron trifluoride diethyl etherate, isobutyryl chloride and parafor-
maldehyde were purchased from Nacalai Tesque. b-Cyanoethyl 2’-
O-TBDMS RNA phosphoramidites, Glen UnySupport 500 and other
reagents for oligonucleotide synthesis were purchased from Glen
Research. Solid supports (dT-CPG 500 and 5’-DMT-Suc-CPG 500)
loaded with dT were purchased from Glen Research and Biosearch
technologies. Sep-Pak column (tC18 pPlus short cartridge, 37–
55 mm) was purchased from Waters. Natural 21-mer oligonucleo-
tides were purchased from Hokkaido System Science, and 4-(2-hy-
droxyethyl)-1-piperazineethanesulfonic acid-potassium hydroxyde
(HEPES-KOH) buffer was purchased from JBioS. Fetal bovine serum
(FBS) was purchased from BioWest. pEYFP-N1 plasmid, Prime Script
RT reagent Kit (Perfect Real Time) and Premix Ex Taq were pur-
chased from Takara, and pTagRFP-C was purchased from Evrogen.
Lipofectamine 2000 and TURBO DNase were purchased from Life
Technologies. ISOGEN was purchased from NIPPON GENE. Phos-
phate-buffered saline (PBS) was purchased from Nissui. Fluores-
cence mounting medium was purchased from DAKO.
Quantification of RNAi activity with RT-PCR
Gene expression in terms of mRNA transcripts was quantified by
quantitative RT-PCR, and the RNAi activity was evaluated as an in-
terfered level of the EYFP expression that was quantified relative to
the tagRFP expression as an internal reference.[17] The relative ex-
pression value was further standardized by the value from the neg-
ative control (see above) to afford the EYPF/tagRFP value in per-
centage (Figure 3). The total RNA was extracted from HeLa cells
using ISOGEN (200 mL) and was treated with DNase (TURBO DNase,
4 U). The total RNA (0.5 mg) was transcribed to cDNA using the Pri-
meScript RT reagent kit (Perfect Real Time), and 1/25th of cDNA so-
lution was subjected to PCR with PremixEx Taq (10 mL) in the pres-
ence of a primer (0.2 mm) and a TaqMan probe (0.1 mm). For EYFP
analysis, the primers were 5’-d(CpCpApCpTpApCpCpApGpCpApG-
pApApCpApCpCpC)-3’
d(CpTpCpGpTpTpGpGpGpGpTpCpTpTpTpGpCpTpCpApG)-3’, and
the TaqMan probe was 5’-FAM-
d(TpGpCpTpGpCpTpGpCpCpCpGpApCpApApCpCpApCpT-
and
5’-
pApCpCpT)-TAMRA-3’. For tagRFP analysis, the primers were 5’-
d(CpTpGpGpCpTpApCpCpApGpCpTpTpCpApTpGpTpApCpG)-3’
and 5’-d(CpCpCpTpCpApGpGpGpApApGpGpApCpTpGpCpTpTpA)-
3’, and the TaqMan probe was 5’-FAM-d(ApGpCpApGpA-
pApCpCpTpTpCpApTpCpApApCpCpApCpApCpCpCpApGpG)-
TAMRA-3’. The PCR was carried out, after heat treatment at 958C
for 3 min, by 40 cycles of 958C for 15 s and 608C for 1 min on an
ABI 7300 Real Time PCR System. The RNAi experiments were car-
ried out at least in duplicate, and the average values are shown in
Figure 3.
Qualitative analysis of RNAi activity with fluorescent micros-
copy
Synthesis
Gene expression in terms of translated proteins was imaged by flu-
orescent microscopy. The siRNA experiments were performed on
a cover glass embedded in the well for translocation. The cells
were washed with PBS, fixed with 4% v/v paraformaldehyde/PBS
for 15 min and stained with DAPI (0.2 mgmLÀ1 in PBS) for 10 min.
The slide glass was wetted with fluorescence mounting medium,
and the cover glass with cells was translocated. Marker proteins of
EYFP and tagRFP were imaged with an exposure time of 0.5 s, and
DAPI stains were imaged with an exposure time of 0.05 s.
See the Supporting Information for the procedures and spectral
data.
Formation of siRNA duplex for RNAi
A mixture of a passenger strand 7 (20 mm) and a guide strand 8
(20 mm) in HEPES-KOH buffer (30 mm, pH 7.4) containing potassium
acetate (100 mm) and magnesium acetate (10 mm) was heated at
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Acknowledgements
This work was partly supported by KAKENHI (26810083).
Keywords: click chemistry · oligonucleotides · RNA · siRNA ·
solid-phase synthesis
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Manuscript received: July 22, 2015
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Accepted Article published: August 6, 2015
Final Article published: August 26, 2015
Chem. Asian J. 2015, 10, 2683 – 2688
www.chemasianj.org
2688
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