Amido-bridged nucleic acids with small hydrophobic residues enhance hepatic tropism of antisense oligonucleotides in vivo

Article abstract of DOI:10.1039/c5ob00242g

High scalability of a novel bicyclic nucleoside building block, amido-bridged nucleic acid (AmNA), to diversify pharmacokinetic properties of therapeutic antisense oligonucleotides is described. N2′-functionalization of AmNA with a variety of hydrophobic groups is straightforward. Combinations of these modules display similar antisense knockdown effects and improve cellular uptake, relative to sequence-matched conventional 2′,4′-bridged nucleic acid (2′,4′-BNA) in vivo. This journal is

Full text of DOI:10.1039/c5ob00242g

Organic &
Biomolecular Chemistry
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Amido-bridged nucleic acids with small
hydrophobic residues enhance hepatic tropism
of antisense oligonucleotides in vivo†
Cite this: Org. Biomol. Chem., 2015,
13, 3757
Tsuyoshi Yamamoto,‡a,b Aiko Yahara,‡a Reiko Waki,a Hidenori Yasuhara,a,b
Fumito Wada,a,b Mariko Harada-Shibab and Satoshi Obika*a
High scalability of a novel bicyclic nucleoside building block, amido-bridged nucleic acid (AmNA),
to diversify pharmacokinetic properties of therapeutic antisense oligonucleotides is described.
N2’-functionalization of AmNA with a variety of hydrophobic groups is straightforward. Combinations of
these modules display similar antisense knockdown effects and improve cellular uptake, relative to
sequence-matched conventional 2’,4’-bridged nucleic acid (2’,4’-BNA) in vivo.
Received 4th February 2015,
Accepted 5th February 2015
DOI: 10.1039/c5ob00242g
www.rsc.org/obc
this class of nucleotides are basically formidable. Therefore,
evaluating the pharmacokinetics of AONs is formidable as well
Introduction
It has recently been demonstrated that naked therapeutic anti- because we have only the option of evaluating them in vivo. On
sense oligonucleotides (AONs) exhibit robust systemic activity the other hand, Leumann et al. introduced hydrophobic side
when comprised of several chemically modified nucleic acid chains into the C6′-position of each tricyclo-DNA monomer
building blocks.1 In particular, conformationally constrained through a metabolically labile ester group, then integrated
nucleotides such as 2′,4′-bridged nucleic acid (2′,4′-BNA) [also these nucleotides into oligonucleotides and successfully
known as locked nucleic acid (LNA)]2 in combination with improved cell-membrane permeability.8 However, the effect of
phosphorothioate (PS) exhibit extraordinarily high target RNA integration of these monomers on in vivo deposition remains
binding and acceptable pharmacokinetics. However, only a obscure. In this context, we recently reported a promising
small fraction of the administered PS–LNAs is distributed in alternative scaffold nucleoside, amido-bridged nucleic acid
the target tissues;3 most of the dose is deposited subcellularly, (AmNA),9 which may be useful for addressing this issue
which is undesirable.4 Thus, overcoming the pharmacokinetic (Fig. 1).
challenges of AONs is necessary to improve their potency and
to address safety concerns.5
The furanose of AmNA is fused to a five-membered
γ-lactam, whose amide bond is bridged between C2′ and C4′ of
A number of targeted delivery strategies for antisense thera- the ribose and rigidly fixed in C3′-endo conformation. The
peutics have been developed, including the terminal conju- AmNA-modified AONs are much less susceptible to nuclease
gation of biofunctional molecules.6 However, these ligands digestion than their LNA counterparts, possibly because of the
often interfere with knockdown activity, despite their advan- steric hindrance of the N2′-methyl and neighboring carbonyl
tageous effect on the pharmacokinetics of AONs. In contrast, groups of the amide. These modified AONs maintain LNA-like
numerous LNA analogues with unique bridging structures high RNA affinity and show higher in vitro antisense activity
have been developed and refined, and apparently minor struc- than their LNA counterparts. Notably, N2′-functionalization is
tural modification of LNA can significantly alter their bio- usually facile (AmNA[N-R]), making AmNA a promising build-
logical properties.3a,7 However, the design and synthesis of ing block for AONs. Other multivalent heteroatom-containing
aGraduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka,
Suita, Osaka 565-0871, Japan. E-mail: obika@phs.osaka-u.ac.jp;
Fax: +81 6 6879 8204; Tel: +81 6 6879 8200
bDepartment of Molecular Innovation in Lipidology, National Cerebral and
Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565,
Japan
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5ob00242g
Fig. 1 Tandem arrangement strategy of N2’-functionalized AmNAs for
‡These authors contributed equally to this work.
improving the pharmacokinetics of antisense therapeutics.
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BNAs, such as 2′-amino-LNA,3c,10 2′,4′-BNANC,7f N-Me- sodium hydride, followed by the addition of the corresponding
aminooxy BNA7c and 6′-thiol containing BNA,11 are also intri- alkyl halides to give N-substituted compounds 2a–g. Despite
guing alternatives in the same context, but N- or S-functiona- steric hindrance, these coupling reactions provided high yields
lized derivatives remain to be comprehensively developed.
under optimized conditions. Hydrogenolysis of the O3′, O5′-
To demonstrate the plasticity of our scaffold nucleoside for benzyl groups as well as N3-benzyloxymethyl groups of 2a–g
improving antisense therapeutics, we have developed a meth- was effected using palladium on the carbon catalyst in THF
odology to perturb the “in vivo” pharmacokinetics of AONs by followed by O5′-dimethoxytritylation to give N-substituted
using a variety of AmNA derivatives. Specifically, we syn- monomers 3a–g in good yield over two steps (52–96%).
thesized AmNAs functionalized with a series of hydrophobic Subsequent O3′-phosphitylation of 3a–g was achieved with
groups for potential improvement of intracellular and hepatic 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite
uptake of AONs and showed that hydrophobicity of the provide desired thymine phosphoramidites 4a–g.
AmNA-AONs is readily adjustable by altering the substituents.
to
Oligonucleotide synthesis
We also demonstrated that a better tandem combination of
N-alkylated AmNA (AmNA[N-R]) modules can improve hepatic
disposition and potency of AONs in vivo.
Synthesis of oligonucleotides (ONs) containing AmNA[N-R]
monomers was performed on an automated DNA synthesizer
using a conventional phosphoramidite method. 5-[3,5-Bis(tri-
fluoromethyl)phenyl]-1H-tetrazole (Activator 42®) solution was
used for the synthesis of all the oligonucleotides described
here. N-Alkylated AmNAs 4a–g were successfully coupled using
an extended coupling time to 16 min. The synthesized ONs
were purified by reverse-phase HPLC (RP-HPLC) and the com-
position and purity were analyzed by MALDI-TOF mass
spectrometry and RP-HPLC, respectively (Table 1 and ESI
Table S1†). A purity greater than 95% was confirmed for all oligo-
nucleotides. Note that the large-scale synthesis of phosphoro-
thioate antisense oligonucleotides for in vivo usage and their
purification were conducted by Gene Design Inc. (Ibaraki,
Japan), where they used AmNA[N-R] phosphoramidite mono-
mers provided by us and the composition and purity were
analyzed by MALDI-TOF mass spectrometry and RP-HPLC,
respectively.
Results and discussion
Phosphoramidite monomer synthesis
The phosphoramidite monomers corresponding to AmNA-
[N-H] 4a and AmNA[N-Me] 4b were obtained as previously
described.9 From known nucleoside 1, we synthesized the five
monomers AmNA[N-Et] 4c, AmNA[N-nPr] 4d, AmNA[N-iPr] 4e,
AmNA[N-Bn] 4f and AmNA[N-Phen] 4g, where abbreviated sub-
stituents indicate methyl, ethyl, n-propyl, i-propyl, benzyl and
phenethyl, respectively (Scheme 1). Briefly, 1 was treated with
Physicochemical properties of AmNA-containing
oligonucleotides
We compared the relative hydrophobicity of oligonucleotides
singly modified with AmNAs (4a–g; ON-1–8) by RP-HPLC using
an octadecyl (C18) silica column under the indicated con-
ditions. The obtained retention times are shown in Table 1.
AmNA[N-H]-modified oligonucleotide ON-2 was the most
Table 1 Oligonucleotides singly modified with AmNAsa
Tm (ΔTm/mod.)
(°C)
Retention
Oligonucleotides
ID
RNA
DNA
time (min)b
5′-d(TTTTTTTTTT)-3′
5′-d(TTTTTtHTTTT)-3′
5′-d(TTTTTtMTTTT)-3′
5′-d(TTTTTtETTTT)-3′
5′-d(TTTTTtnTTTT)-3′
5′-d(TTTTTtiTTTT)-3′
5′-d(TTTTTtBTTTT)-3′
5′-d(TTTTTtPTTTT)-3′
ON-1
ON-2
ON-3
ON-4
ON-5
ON-6
ON-7
ON-8
19
21
8.4
7.1
26 (+7)
26 (+7)
24 (+5)
21 (+2)
22 (+3)
23 (+4)
21 (+2)
20 (−1)
18 (−3)
18 (−3)
18 (−3)
17 (−4)
18 (−3)
18 (−3)
10.7
13.5
15.5
17.0
22.4
25.8
Scheme 1 Reagents and conditions: (i) NaH, RX, DMF, 0 °C → rt, R = Et:
EtBr, quant.; R = nPr: nPrBr, 76%; R = iPr: iPrI, quant.; R = Bn: BnBr,
quant.; R = CH2CH2Ph: BrCH2CH2Ph, 43%; (ii) 20% Pd(OH)2/C, H2, THF,
rt; (iii) DMTrCl, pyridine, rt, R = Et: 74% (2 steps); R = nPr: 96% (2 steps);
R = iPr: 81% (2 steps); R = Bn: 90% (2 steps); R = CH2CH2Ph: 83%
(2 steps); (iv) (i-Pr2N)2POCH2CH2CN, N,N-diisopropylammonium tetra-
zolide, MeCN/THF (3 : 1), rt, R = Et: 77%; R = nPr: 88%; R = iPr: 49%; R =
Bn: 61%; R = CH2CH2Ph: 48%.
a tH = AmNA[N-H], tM = AmNA[N-Me], tE = AmNA[N-Et] , tn = AmNA[N-
nPr], ti = AmNA[N-iPr], tB = AmNA[N-Bn], and tP = AmNA[N-Phen].
b Conditions: eluent A: 0.1 M TEAA buffer, eluent B: A/MeCN (1/1, v/v),
gradient: MeCN conc. = 8–13% (30 min), 260 nm.
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Table 2 Antisense oligonucleotides targeting murine apoC-III mRNAa
hydrophilic. As expected, the retention times of the derivatives
varied as a function of the hydrophobicity of the oligonucleo-
tides and could be adjusted by changing the substituents and
their number.
Tm (ΔTm/
mod.)
(°C)
RNA
Retention
Oligonucleotides
ID
time (min)b
To estimate the effect of modifications (4a–g) on duplex
stability, the thermal stability of duplexes was measured with
unmodified complementary RNA and DNA strands and com-
pared with the melting temperatures of the corresponding
unmodified reference duplexes (Table 1). Single incorporation
of AmNAs 4a–g into the center of a DNA T decamer increased
thermal stability for complementary RNA (ΔTm = +2 to +7 °C),
but decreased stability for complementary DNA (ΔTm = −1 to
−4 °C), indicating that high RNA-selective binding is main-
tained. These results are consistent with previous observations
made using a series of BNAs whose bridges differed in size
and composition from LNA.7a,12 A previous crystal structure
study of DNA–LNA heteroduplex revealed that the 2′-oxygen of
LNA forms hydrogen bonds with water molecules.13 The per-
turbation of these hydration patterns by the 2′-substituents of
AmNAs probably affected duplex stability, but all derivatives
retained high affinity.
5′-d(TTATCCAGCTTTATTA)-3′
5′-d(tLtLAtLCCAGCTTtLAtLtLA)-3′
5′-d(tMtMAtMCCAGCTTtMAtMtMA)-3′ ON-11S 63 (+4)
5′-d(titiAtiCCAGCTTtiAtitiA)-3′
5′-d(tBtBAtBCCAGCTTtBAtBtBA)-3′
5′-d(titiAtMCCAGCTTtMAtitiA)-3′
5′-d(tBtMAtMCCAGCTTtMAtMtBA)-3′
ON-9S
ON-10S 63 (+4)
39
5.8
5.8
5.8
10.6
21.2
11.0
13.0
ON-12S 61 (+4)
ON-13S 59 (+3)
ON-14S 62 (+4)
ON-15S 63 (+4)
a tL = LNA-T, tM = AmNA[N-Me], ti = AmNA[N-iPr] and tB = AmNA[N-Bn].
b Conditions: eluent A: 0.1 M TEAA buffer, eluent B: A/MeCN (1/1, v/v),
gradient: MeCN conc. = 13–37% (30 min), 260 nm.
tive hydrophobicity of the oligonucleotides was gauged from
their elution time of RP-HPLC.
Each PS modification increases the retention time of the
AON; consequently the content of acetonitrile in the elution
buffer was modified from 8–13% to 13–37%, as described in
the footnote to Table 2. The retention times for ON-11S to -13S
differed significantly and predictably from the singly-incorpor-
ated phosphodiester-version oligonucleotides (ON-3, -6, -7;
Table 1). AmNA[N-Bn] 4f and AmNA[N-iPr] 4e increased the
hydrophobicity of the AON, whereas steric hindrance of the
gap moiety in ON-12S and -13S reduced potency. Therefore, we
further designed and synthesized ON-14S and -15S, which
showed well-controlled retention times. The Tm values of
ON-11S to -15S were measured and found to be in approxi-
mately 60 °C under the indicated buffer conditions. C57Bl/6J
male mice (n = 3 per group) were intravenously injected with
ON-10S, -11S, -12S, -13S, -14S or -15S at a dose of 2.9 µmol
kg−1 (15 mg kg−1 for ON-10S). LNA counterpart ON-10S
reduced apoC-III by 45%, and AmNA-AONs ON-11S and
ON-12S achieved a similar knockdown of 40% and 30%
respectively. This is the first demonstration of AmNA-AONs
exhibiting LNA congener-like high activity in vivo (Fig. 2, ESI
Fig. S8†). In contrast, AmNA[N-Bn] 4f-based AON ON-13S
showed no knockdown. The less hindered ON-14S and -15S in
the gaps showed improved potency compared to -11S, -12S;
interestingly, ON-14S achieved the highest knockdown of
apoC-III mRNA of the AmNA-AONs tested, suggesting that a
combination of functionalized AmNAs working in tandem can
tailor potency of AONs.
Nuclease stability
The effect of AmNA modification on nuclease resistance was
determined by incorporating AmNAs into the second base
from the 3′-end of oligonucleotides, followed by incubation
with CAPV for 40 min at 37 °C. The percentage of intact oligo-
nucleotides was analyzed by RP-HPLC and found to be higher
than with conventional AmNA[N-Me] (ESI Fig. S5†).7a
In vivo activities of AmNA-modified antisense oligonucleotides
We next evaluated hydrophobic AmNA-modified AONs in vivo.
We previously developed a potent LNA-based AON that targets
apolipoprotein CIII (apoC-III) for the treatment of dyslipide-
mia.14 Truncated versions of this apoC-III AON were used in
these in vivo studies because we recently revealed that these
could be more potent.3d,15 Six LNAs were incorporated into a
16-mer PS-AON, ON-9S. ON-10S retains a seven natural-nucleo-
tide gap moiety, sufficient for maintaining RNase H (a key
enzyme for antisense mechanism)-recruiting activity.
The primary purpose of the in vivo study was to confirm the
effectiveness of this truncated version of LNA-AON to target
apoC-III mRNA, and mice were dosed intravenously with
ON-10S at a dose range of 5–20 mg kg−1. The expression levels
of apoC-III mRNA in the liver were analysed 72 hours post-
injection. Dose-dependent reduction in hepatic apoC-III
Hepatic tropism of hydrophobic AmNA-modified antisense
oligonucleotides
mRNA through
a single administration of ON-10S was
observed without significant toxicity (ESI Fig. S6, S7.† The
highest reduction in hepatic apoC-III mRNA (60%) was To investigate whether different combinations of hydrophobic
recorded at a dose of 20 mg kg−1, and statistical significance AmNAs alter the tissue deposition of AONs, we measured the
with saline control was found at doses above 10 mg kg−1
.
intact AONs that accumulated in the liver after intravenous
Thus, hydrophobic AmNAs-carrying AONs were evaluated administration using a previously described ELISA method.16
in vivo at 15 mg kg−1
The hepatic distribution of the more hydrophobic ON-12S,
.
N-Alkylated AmNAs 4b, e, f were introduced into PS-AON -13S, -14S and -15S was ∼1.5 times higher than that of the
ON-9S to obtain ON-11S to -13S as shown in Table 2. The rela- LNA-AON ON-10S, whereas ON-10S and ON-11S exhibited
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Conclusions
In summary, we here showed that AmNAs are an interesting
class of antisense building blocks. A series of AmNAs functio-
nalized with a variety of hydrophobic groups were synthesized:
AmNA[N-Et] 4c, AmNA[N-nPr] 4d, AmNA[N-iPr] 4e, AmNA-
[N-Bn] 4f and AmNA[N-Phen] 4g. We demonstrated that a
tandem arrangement of these small substituents affects the
systemic activity and tissue disposition of AONs in vivo. This
strategy will allow more finely-tuned control of the properties
of AONs than conventional strategies. Using this scaffold
nucleoside, we will further develop useful modules and ident-
Fig. 2 Reduction of apoC-III mRNA in the livers of mice receiving a ify the best combination of these AmNA modules to tackle the
single intravenous dose of 2.868 µmol kg−1 of a series of AONs. Dun-
nett’s multiple comparison test, ***P < 0.001, N.S.; not significant. Error
bars represent group means + S.D. n = 3.
issues currently confronting antisense therapeutics.
Experimental
General
All moisture-sensitive reactions were carried out in well-dried
glassware under
a N2 atmosphere. Anhydrous dichloro-
methane, DMF, MeCN, and pyridine were used as purchased.
1H NMR spectra were recorded at 300 and 400 MHz and
500 MHz, 13C NMR were recorded at 75 and 100 MHz, and the
31P spectrum was recorded at 161 MHz. Chemical shift values
are expressed in δ values (ppm) relative to tetramethylsilane
(TMS) as an internal standard and a residual solvent for 1H
NMR, and CHCl3 (δ = 77.00 ppm), methanol (δ = 49.00 ppm),
and DMSO (39.50 ppm) for 13C NMR, and 85% H3PO4 (δ =
0 ppm) for 31P NMR. Fast atom bombardment mass spectra
(FAB-MS) were recorded in the positive ion mode. For column
chromatography, silica gel PSQ 100B was used. Progress of the
reaction was monitored by analytical thin layer chromato-
graphy (TLC) on pre-coated aluminium sheets (Silica gel
60 F254 sheet, Merck), and the products were visualized by UV
light.
Fig. 3 ELISA-based quantification of a series of AONs distributed in
murine liver after 72 hours post-injection. Error bars represent group
means + S.D. n = 3.
hepatic distribution similar to each other (Fig. 3). However,
the activity of all these AmNA-based AONs was at best compar-
able to the LNA counterpart ON-10S. It should primarily be
noted that no statistical significance was found between these
liver AON contents, which may contribute to this insignificant
change in potency. It is also possible that the hydrophobic
modification altered the suborgan distribution of AONs in
liver. ApoC-III mRNA is predominantly expressed in hepatic
parenchymal cells, which comprise 80% of the liver volume,
whereas non-parenchymal cells such as Kupffer cells and sinu-
soidal endothelial cells are relatively minor components of the
liver. AONs should therefore selectively target the parenchymal
cells. However, it is reported that 80% of PS-AONs accumulated
in the liver are distributed in non-parenchymal cells and only
20% are in the parenchymal hepatocytes.4b The hydrophobic
AONs developed here might foster this trend. Alternatively,
there are at least two uptake pathways in parenchymal cells
(a productive pathway and a bulk nonproductive pathway); the
hydrophobic AONs might encourage the nonproductive uptake
which undermines the knockdown activity of AONs.4a
Synthesis of AmNA monomers and phosphoramidites
General procedure 1 (synthesis of compound 2). To the stir-
ring solution of 1 (1.0 equiv.) in DMF (0.1 M) was added NaH
(1.2 equiv.) at 0 °C. After stirring for 30 min, alkyl halide
(1.2 equiv.) was added. The reaction temperature was gradually
raised from 0 °C to room temperature and after completion of
the reaction (approx. 30 min), ice-cold water was added. The
solution was stirred for 15 min and the product was extracted
with ethyl acetate. The organic phase was washed with brine,
dried (Na2SO4), and concentrated. The product was purified
by flash column chromatography (n-hexane–ethyl acetate =
3 : 1 or 2 : 1) to afford 2 as a white amorphous solid.
(2′R)-3′,5′-Di-O-benzyl-N3-benzyloxymethyl-2′-ethylamino-2′-N,4′-
C-oxomethylenethymidine (2c: R = Et). By following the general
procedure 1, using bromoethane as an alkyl halide, 2c was
obtained in quant. as a white amorphous solid.
[α]2D3 +45.9 (c 0.100, CHCl3). IR (KBr): 3071, 3033, 2930,
1
2871, 1725, 1708, 1665, 1454, 1273 cm−1. H NMR (300 MHz,
CDCl3) δ: 1.22 (3H, t), 1.64 (3H, s), 3.35 (1H, dq J = 14 Hz,
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7 Hz), 3.52 (1H, dq J = 14 Hz, 7 Hz), 3.95, 4.10 (2H, AB, J = HRMS (FAB): calcd for C40H40N3O7 [M + H]+: 674.2866. Found:
11.5 Hz), 4.07 (1H, s), 4.13 (1H, s), 4.52, 4.54 (2H, AB, J = 674.2841.
(2′R)-3′,5′-Di-O-benzyl-N3-benzyloxymethyl-2′-phenethylamino-
12 Hz), 4.59, 4.63 (2H, AB, J = 11.5 Hz), 4.70 (2H, s), 5.39 (1H,
2′-N,4′-C-oxomethylenethymidine (2g: R = CH2CH2Ph). By follow-
ing the general procedure 1, using 2-bromoethylbenzene as an
alkyl halide, 2g was obtained in 43% as a white amorphous
solid.
s), 5.45, 5.47 (2H, AB, J = 9.5 Hz), 7.18–7.39 (15H, m), 7.54 (1H,
s). 13C NMR (100 MHz, CDCl3) δ: 12.8, 13.7, 36.5, 62.4, 63.2,
70.1, 72.2, 72.3, 73.7, 76.7, 78.1, 85.4, 87.6, 109.8, 127.6, 127.6,
127.9, 128.1, 128.4, 133.8, 136.3, 137.4, 137.6, 150.5, 163.1,
170.1. MS (FAB): m/z 612 [M + H]+. HRMS (FAB): calcd for
C35H38N3O7 [M + H]+: 612.2710. Found: 612.2693.
[α]2D4 +0.2 (c 0.320, CHCl3). IR (KBr): 3029, 2930, 1722, 1701,
1667, 1453, 1274 cm−1. 1H NMR (300 MHz, CDCl3) δ: 1.62 (3H,
s), 2.82–3.02 (2H, m), 3.52–3.75 (2H, m), 3.94, 4.08 (2H, AB, J =
(2′R)-3′,5′-Di-O-benzyl-N3-benzyloxymethyl-2′-propylamino-2′-
N,4′-C-oxomethylenethymidine (2d: R = nPr). By following the 12 Hz), 4.02 (1H, s), 4.12 (1H, s), 4.44 (2H, s), 4.58, 4.63 (2H,
general procedure 1, using 1-bromopropane as an alkyl halide, AB, J = 11.5 Hz), 4.70 (2H, s), 5.29 (1H, s), 5.44, 5.48 (2H, AB,
2d was obtained in 76% as a white amorphous solid.
J = 9.5 Hz), 7.14–7.40 (20H, m), 7.49 (1H, s). 13C NMR
[α]2D3 +29.2 (c 1.000, CHCl3). IR (KBr): 3066, 3029, 2910, (100.53 MHz, CDCl3) δ: 13.0, 34.7, 43.1, 63.3, 63.3, 70.3, 72.4,
1
2863, 1724, 1709, 1664, 1455, 1274 cm−1. H NMR (300 MHz, 72.5, 74.0, 78.3, 85.3, 87.5, 110.0, 126.7, 127.7, 127.7, 127.8,
CDCl3) δ: 0.93 (3H, q, J = 7.0 Hz), 1.56–1.64 (2H, m), 1.64 (3H, 128.1, 128.3, 128.3, 128.6, 128.6, 128.7, 133.9, 136.4, 137.5,
s), 3.33 (2H, ddq), 3.95, 4.09 (2H, AB, J = 12 Hz), 4.07 (1H, s), 137.8, 137.9, 150.6, 163.3, 170.7. MS (FAB): m/z 688 [M + H]+.
4.13 (1H, s), 4.53 (2H, s), 4.59, 4.65 (2H, AB, J = 11.0 Hz), 4.71 HRMS (FAB): calcd for C41H42N3O7 [M + H]+: 688.3023. Found:
(2H, s), 5.40 (1H, s), 5.46 (2H, t, J = 9.5 Hz), 7.19–7.37 (15H, 688.3015.
m), 7.54 (1H, s). 13C NMR (100 MHz, CDCl3) δ: 11.1, 12.9, 21.9,
General procedure 2 (synthesis of compound 3). To the
43.4, 62.9, 63.3, 70.2, 72.3, 72.3, 72.4, 73.8, 78.2, 85.3, 87.5, solution of 2 (1.0 equiv.) in THF or methanol (0.1 M) was
109.8, 127.6, 127.6, 127.6, 127.7, 128.0, 128.2, 128.2, 128.2, added 20% palladium on carbon (1.0 w/w) and the reaction
128.4, 133.8, 136.3, 137.4, 137.4, 137.7, 150.6, 163.2, 170.5. MS vessel was degassed several times with hydrogen. The reaction
(FAB): m/z 626 [M + H]+. HRMS (FAB): calcd for C36H40N3O7 mixture was stirred under a hydrogen atmosphere for 1–5 h at
[M + H]+: 626.2866. Found: 626.2867.
room temperature. After completion of the reaction, the reac-
tion solution was filtered using filter paper and washed
thoroughly with hot methanol. After evaporation of solvents,
the product was dissolved with methanol (0.3 M) and 28%
ammonia solution (0.3 M) was added and the solution was
stirred at room temperature. After 5 min, the product was con-
centrated to afford S1 as a white solid.
(2′R)-3′,5′-Di-O-benzyl-N3-benzyloxymethyl-2′-isopropylamino-
2′-N,4′-C-oxomethylenethymidine (2e: R = iPr). By following the
general procedure 1, using 2-iodopropane as an alkyl halide,
2e was obtained in quant. as a white amorphous solid.
[α]2D3 +57.2 (c 1.000, CHCl3). IR (KBr): 3064, 3031, 2927,
2873, 1728, 1708, 1666, 1455, 1275 cm−1. H NMR (400 MHz,
1
CDCl3) δ: 1.18 (3H, d, J = 7 Hz), 1.31 (3H, d, J = 7 Hz), 1.64 (3H,
s), 3.94, 4.10 (2H, AB, J = 12 Hz), 4.07 (1H, s), 4.28 (1H, s),
4.36–4.47 (1H, m), 4.50, 4.54 (2H, AB, J = 11.5 Hz), 4.58, 4.63
(2H, AB, J = 11.5 Hz), 4.71 (2H, s), 5.37 (1H, s), 5.46, 5.49 (2H,
AB, J = 9.5 Hz), 7.18–7.39 (15H, m), 7.54 (1H, s). 13C NMR
(100.53 MHz, CDCl3) δ: 12.9, 20.6, 21.1, 42.6, 58.9, 63.2, 70.1,
72.2, 72.2, 73.7, 78.2, 86.1, 88.0, 109.6, 127.3, 127.4, 127.5,
127.6, 127.6, 127.9, 128.1, 128.2, 128.2, 128.4, 129.4, 133.8,
136.2, 137.4, 137.7, 150.5, 163.2, 169.4. MS (FAB): m/z 626
To the solution of S1 in anhydrous pyridine (0.1 M) was
added DMTrCl (1.3 equiv.) and the solution was stirred at
room temperature. After stirring for 1–19 h, ice-cold water was
added and the product was extracted with ethyl acetate. The
organic phase was washed with brine, dried (Na2SO4), and con-
centrated. The product was purified by flash column chrom-
atography (n-hexane–ethyl acetate = 1 : 1 or 1 : 3) to afford 3 as
a white amorphous solid.
(2′R)-5′-O-(4,4′-Dimethoxytrityl)-2′-ethylamino-2′-N,4′-C-oxo-
[M + H]+. HRMS (FAB): calcd for C36H40N3O7 [M + H]+: methylenethymidine (3c: R = Et). By following the general pro-
626.2866. Found: 626.2869.
cedure 2, 3c was obtained in 45% (for 2 steps) as a white
amorphous solid.
(2′R)-3′,5′-Di-O-benzyl-N3-benzyloxymethyl-2′-benzylamino-2′-
N,4′-C-oxomethylenethymidine (2f: R = Bn). By following the
general procedure 1, using bromomethylbenzene as an alkyl
halide, 2f was obtained in quant. as a white amorphous solid.
[α]2D3 +17.1 (c 1.000, CHCl3). IR (KBr): 3064, 3030, 2871,
[α]2D5 +12.9 (c 1.000, CHCl3). IR (KBr): 3087, 2967, 2872,
1
1729, 1698, 1665, 1509, 1464 cm−1. H NMR (300 MHz, CDCl3)
δ: 1.16 (3H, t, J = 7 Hz), 1.62 (3H, s), 3.23–3.55 (2H, m), 3.63,
3.84 (2H, AB, J = 11.5 Hz), 3.74 (6H, s), 4.10 (1H, s), 4.24 (1H,
s), 4.51 (1H, s), 5.39 (1H, s), 6.79–6.83 (4H, m), 7.13–7.45 (9H,
m), 7.81 (1H, s), 10.33 (1H, s). 13C NMR (100.53 MHz, CDCl3)
δ: 12.4, 13.4, 36.5, 55.1, 56.3, 64.9, 72.6, 85.3, 86.6, 88.8, 110.5,
112.9, 113.2, 127.0, 127.6, 127.8, 127.9, 129.0, 130.0, 135.1,
135.4, 144.3, 150.2, 158.4, 164.6, 170.5. MS (FAB): m/z 636
[M + Na]+. HRMS (FAB): calcd for C34H35N3O8Na [M + Na]+:
636.2322. Found: 636.2312.
1728, 1665, 1454, 1277 cm−1 1H NMR (300 MHz, CDCl3) δ:
.
1.60 (3H, s), 3.98, 4.12 (2H, AB, J = 12 Hz), 4.20, 4.26 (2H, AB,
J = 11 Hz), 4.21, 4.85 (2H, AB, J = 15 Hz), 4.58, 4.65 (2H, AB, J =
11.5 Hz), 4.67 (2H, s), 5.34 (1H, s), 5.39, 5.45 (2H, AB, J =
9.5 Hz), 7.02–7.36 (20H, m), 7.48 (2H, s). 13C NMR (100 MHz,
CDCl3) δ: 13.0, 45.1, 61.9, 63.2, 70.3, 72.3, 72.4, 74.0, 77.8,
85.0, 87.5, 109.9, 127.5, 127.7, 127.8, 128.0, 128.1, 128.1, 128.3,
128.4, 128.6, 128.6, 128.8, 133.9, 135.8, 136.2, 137.5, 137.8,
(2′R)-5′-O-(4,4′-Dimethoxytrityl)-2′-propylamino-2′-N,4′-C-oxo-
140.0, 150.5, 151.6, 163.3, 170.5. MS (FAB): m/z 674 [M + H]+. methylenethymidine (3d: R = nPr). By following the general
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procedure 2, 3d was obtained in 96% (for 2 steps) as a white 137.9, 150.6, 163.3, 170.7. MS (FAB): m/z 712 [M + Na]+. HRMS
amorphous solid.
(FAB): calcd for C40H39N3O8Na [M + Na]+: 712.2635. Found:
[α]2D6 +33.1 (c 1.000, CHCl3). IR (KBr): 3384, 3021, 2838, 712.2631.
1704, 1699, 1509, 1254 cm−1 1H NMR (300 MHz, CDCl3) δ:
.
General procedure 3 (synthesis of compound 4). To the
solution of 3 (1.0 equiv.) in anhydrous MeCN–THF (3 : 1,
0.1 M) were added N,N-diisopropylammonium tetrazolide
(0.75 equiv.) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphos-
phorodiamidite (1.2 equiv.). After stirring at room
temperature for 9 h–19 h, ice-cold water was added and the
product was extracted with ethyl acetate. The organic
phase was washed with brine, dried (Na2SO4), and concen-
trated. The product was purified by flash column chromato-
graphy (0.05 eq. of triethylamine in n-hexane–ethyl acetate =
1 : 1) to afford 4c (R = Et: 39 mg, 77%) as a white amorphous
solid.
1.17 (3H, t, J = 7 Hz), 1.62 (3H, s), 3.34–3.58 (2H, m), 3.61, 3.90
(2H, AB, J = 12 Hz), 3.91–3.98 (2H, m), 3.88 (6H, s), 4.28 (1H,
s), 4.53 (1H, d, J = 7 Hz), 5.50 (1H, s), 6.83–6.85 (4H, m),
7.23–7.36 (7H, m), 7.44 (2H, d, J = 7 Hz), 7.82 (1H, s), 9.40 (1H,
s). 13C NMR (75 MHz, CDCl3) δ: 11.2, 12.5, 21.7, 43.6, 55.2,
56.5, 65.2, 72.8, 85.2, 86.9, 88.6, 110.7, 113.3, 113.4, 127.1,
127.9, 128.1, 130.0, 135.1, 135.3, 144.3, 150.1, 158.6, 164.3,
170.6. MS (FAB): m/z 650 [M + Na]+. HRMS (FAB): calcd for
C35H37N3O8Na [M + Na]+: 650.2478. Found: 650.2508.
(2′R)-5′-O-(4,4′-Dimethoxytrityl)-2′-isopropylamino-2′-N,4′-C-
oxomethylenethymidine (3e: R = iPr). By following the general
procedure 2, 3e was obtained in 78% (for 2 steps) as a white
amorphous solid.
(2′R)-3′-O-[2-Cyanoethoxy(disopropylamino)phosphino]-5′-O-
(4,4′-dimethoxytrityl)-2′-ethylamino-2′-N,4′-C-oxomethylenethymi-
dine (4c: R = Et). By following the general procedure 3, 4c was
obtained in 77% as a white amorphous solid.
[α]2D3 −3.83 (c 1.000, CHCl3). IR (KBr): 3366, 3190, 2968,
2836, 1704, 1686, 1509, 1249 cm−1. H NMR (300 MHz, CDCl3)
1
M.p. 100–103 °C (CH2Cl2–hexane). 31P NMR (161.83 MHz,
CDCl3) δ: 149.2, 150.3. MS (FAB): m/z 814 [M + H]+. HRMS
(FAB): calcd for C43H53N5O9P [M + H]+: 814.3581. Found:
814.3588.
(2′R)-3′-O-[2-Cyanoethoxy(disopropylamino)phosphino]-5′-O-
(4,4′-dimethoxytrityl)-2′-propylamino-2′-N,4′-C-oxomethylenethymi-
dine (4d: R = nPr). By following the general procedure 3, 4d
was obtained in 88% as a white amorphous solid.
δ: 1.17 (3H, d, J = 6.5 Hz), 1.28 (3H, d, J = 6.5 Hz), 1.69 (3H, s),
2.54 (1H, s), 3.59, 3.92 (2H, AB, J = 12 Hz), 3.77 (6H, s), 4.28
(1H, s), 4.28–4.37 (1H, m), 4.40 (1H, s), 5.37 (1H, s), 6.82–6.84
(4H, m), 7.20–7.36 (7H, m), 7.44 (2H, d, J = 7 Hz), 7.79 (1H, s),
9.65 (1H, s). 13C NMR (100.53 MHz, CDCl3): δ 12.5, 20.5, 21.1,
42.8, 55.2, 56.5, 61.5, 72.9, 86.0, 86.9, 89.2, 110.6, 113.3, 113.4,
127.1, 127.9, 128.1, 130.0, 135.2, 135.3, 144.4, 150.1, 158.6,
164.3, 169.4. MS (FAB): m/z 650 [M + Na]+. HRMS (FAB): calcd
for C35H37N3O8Na [M + Na]+: 650.2478. Found: 650.2490.
(2′R)-5′-O-(4,4′-Dimethoxytrityl)-2′-benzylamino-2′-N,4′-C-oxo-
methylenethymidine (3f: R = Bn). By following the general pro-
cedure 2, 3f was obtained in 90% (for 2 steps) as a white
amorphous solid.
M.p. 83–86 °C (CH2Cl2–hexane). 31P NMR (161.83 MHz,
CDCl3) δ: 149.8, 150.2. MS (FAB): m/z 828 [M + H]+. HRMS
(FAB): calcd for C44H55N5O9P [M + H]+: 828.3737. Found:
828.3745.
(2′R)-3′-O-[2-Cyanoethoxy(disopropylamino)phosphino]-5′-O-
(4,4′-dimethoxytrityl)-2′-isopropylamino-2′-N,4′-C-oxomethyl-
enethymidine (4e: R = iPr). By following the general procedure
3, 4e was obtained in 32% as a white amorphous solid.
M.p. 101–104 °C (CH2Cl2–hexane). 31P NMR (161.83 MHz,
CDCl3) δ: 149.5, 151.3. MS (FAB): m/z 828 [M + H]+. HRMS
(FAB): calcd for C44H55N5O9P [M + H]+: 828.3737. Found:
828.3729.
[α]2D5 +3.8 (c 1.000, CHCl3). IR (KBr): 3063, 2967, 2837, 1686,
1607, 1509, 1249 cm−1. 1H NMR (300 MHz, CDCl3) δ: 1.57 (3H,
s), 3.67, 3.88 (2H, AB, J = 12 Hz), 3.74 (6H, s), 4.21 (1H, s), 4.44
(1H, s), 4.47, 4.52 (2H, AB, J = 15 Hz), 5.13 (1H, s), 6.79–6.83
(4H, m), 7.14–7.34 (7H, m), 7.39–7.44 (2H, d, J = 7 Hz), 7.74
(1H, s), 9.97 (1H, s). 13C NMR (75 MHz, CDCl3): δ 12.5, 45.6,
55.2, 56.5, 65.1, 72.4, 84.7, 86.8, 88.7, 110.6, 113.0, 113.3,
127.1, 127.9, 128.0, 128.7, 128.8, 130.0, 135.1, 135.2, 135.7,
144.3, 150.1, 158.6, 164.4, 170.7. MS (FAB): m/z 698 [M + Na]+.
(2′R)-3′-O-[2-Cyanoethoxy(disopropylamino)phosphino]-5′-O-
(4,4′-dimethoxytrityl)-2′-benzylamino-2′-N,4′-C-oxomethylenethymi-
HRMS (FAB): calcd for C39H37N3O8Na [M + Na]+: 698.2478. dine (4f: R = Bn). By following the general procedure 3, 4f was
Found: 698.2484.
obtained in 61% as a white amorphous solid.
M.p. 99–101 °C (CH2Cl2–hexane). 31P NMR (161.83 MHz,
CDCl3) δ: 150.0, 150.2. MS (FAB): m/z 876 [M + H]+. HRMS
(FAB): calcd for C48H55N5O9P [M + H]+: 876.3737. Found:
876.3735.
(2′R)-3′-O-[2-Cyanoethoxy(disopropylamino)phosphino]-5′-O-
(4,4′-dimethoxytrityl)-2′-phenethylamino-2′-N,4′-C-oxomethyl-
enethymidine (4g: R = CH2CH2Ph). By following the general
procedure 3, 4g was obtained in 48% as a white amorphous
solid.
(2′R)-5′-O-(4,4′-Dimethoxytrityl)-2′-phenethyl amino-2′-N,4′-C-
oxomethylenethymidine (3g: R = CH2CH2Ph). By following the
general procedure 2, 3g was obtained in 84% (for 2 steps) as a
white amorphous solid.
[α]2D1 +4.6 (c 0.400, CHCl3). IR (KBr): 3190, 2958, 2929, 1721,
1694, 1672, 1509, 1270 cm−1 1H NMR (300 MHz, CDCl3) δ:
.
1.67 (3H, d, J = 8.5 Hz), 2.93–3.01 (2H, m), 3.56–3.66 (1H, m),
3.58, 3.87 (2H, AB, J = 12 Hz), 3.81 (7H, m), 4.07 (1H, s), 4.22
(1H, m), 4.34 (1H, s), 5.30 (1H, s), 6.83–7.85 (4H, m), 7.16–7.54
(14H, m), 7.72 (1H, d, J = 8.5 Hz), 8.61 (1H, s). 13C NMR
(75 MHz, CDCl3) δ: 13.0, 34.7, 43.1, 63.3, 63.3, 70.3, 72.4, 72.5,
74.0, 78.3, 85.3, 87.5, 109.9, 126.7, 127.7, 127.7, 127.8, 128.1,
128.3, 128.3, 128.6, 128.6, 128.7, 133.9, 136.4, 137.5, 137.8,
M.p. 98–101 °C (CH2Cl2–hexane). 31P NMR (161.83 MHz,
CDCl3) δ: 149.6, 150.8. MS (FAB): m/z 890 [M + H]+. HRMS
(FAB): calcd for C49H57N5O9P [M + H]+: 890.3894. Found:
890.3909.
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Paper
Synthesis, purification and characterization of
oligonucleotides
In vivo knockdown study
All animal procedures were performed in accordance with the
Synthesis of 0.2 μmol scale of oligonucleotides ON-6-44 modi- guidelines of the Animal Care Ethics Committee of the
fied with AmNA[N-Me] was performed using Oligonucleotide National Cerebral and Cardiovascular Center Research Insti-
Synthesizer (Gene Design, ns-8) according to the standard tute (Osaka, Japan). All animal studies were approved by an
phosphoramidite protocol with Activator 42™ (Proligo) as the Institutional Review Board. C57BL/6J mice were obtained from
activator. Dry MeCN was used to dissolve AmNA[N-R]. The CLEA Japan. All mice were male, and studies were initiated
standard synthesis cycle was used for assembly of the reagents when animals were 8 weeks of age. Mice (n = 3 per arm) were
and synthesis of the oligonucleotides, except that the coupling maintained on a 12 h light/12 h dark cycle and fed ad libitum.
time was extended to 16 minutes for AmNA[N-R] monomers. Mice were fed a normal chow (CE-2, CLEA Japan) for 2 weeks
(The coupling time for AmNA[N-Me] was 32 seconds.) The syn- before and during treatment. Mice received single treatment of
thesis was carried out in trityl on mode and was treated with saline-formulated AONs intravenously. At the time of sacrifice
concentrated ammonium hydroxide at room temperature for after 72 hours of injection, mice were subjected to blood col-
1 h to cleave the synthesized oligonucleotides from the solid lection from tail veins and then anesthetized with isoflurane
support. The oligonucleotides were initially purified by Sep- (Forane, Abbott Japan) under an overnight fasting condition.
pack Plus C18 Environmental Cartridge. The separated oligonu- Livers were harvested and snap frozen until subsequent analy-
cleotides were further purified by reverse-phase HPLC with sis. Whole blood was collected and subjected to serum separ-
Waters Xbridge™ Shield RP18 2.5 µm (10 mm × 50 mm) ation for subsequent analysis.
columns with a linear gradient of MeCN (7–13% over 42 min
for ON-3–6, ON-11, ON-12, ON-16, 8–15% over 42 min for mRNA quantification
ON-7, ON-8, 13–40% over 42 min for ON-13, ON-17) in 0.1 M
triethylammonium acetate (pH 7.0). The oligonucleotides were
analyzed for purity by HPLC and characterized by MALDI-TOF
mass spectroscopy.
Frozen liver tissue was collected in a 2 mL tube with 1 mL of
TRIzol Reagent (Life Technologies, Japan) and a zirconia ball
(∅ 5 mm, Irie) and mechanically homogenized for 2 min at 30
oscillations per second by a TissueLyser II apparatus (Qiagen).
Total RNA was isolated from the resulting suspensions accord-
ing to the manufacturer’s procedure. Gene expression was eval-
uated by a 2-step quantitative RT-PCR method. Reverse-
transcription of RNA samples was performed by using a High
Capacity cDNA Reverse-Transcription Kit (Applied Biosystems),
and quantitative PCR was performed by TaqMan(R) Fast Uni-
versal PCR Master Mix (Applied Biosystems). The mRNA levels
of target genes were normalized to the GAPDH mRNA level.
For murine apoC-III and GAPDH mRNA quantitation, TaqMan
Gene Expression Assay IDs of Mm00445670_m1 and
Mm99999915_g1 were used.
UV melting experiments and melting profiles
The UV melting experiments were carried out using
a
Shimadzu UV-1650 spectrometer equipped with a Tm analysis
accessory. Equimolecular amounts of the target RNA or DNA
strand and oligonucleotide were dissolved in buffer A (10 mM
phosphate buffer at pH 7.2 containing 100 mM NaCl) to give a
final strand concentration of 4 μM. The samples were annealed
by heating at 95 °C followed by slow cooling to room tempera-
ture. The melting profile was recorded at 260 nm from 0 to
70 °C (for ON-3–8), from 5 to 100 °C (for ON-11–13, 16, 17) at
a scan rate of 0.5 °C min−1. The Tm was calculated as the temp-
erature of the half-dissociation of the formed duplexes, deter-
mined by the midline of the melting curve.
ELISA method for AON quantification in liver
Materials and reagents. The template DNA was a 25-mer
DNA (5′-gaa tag cga taa taa agc tgg ata a-3′), which is comp-
lementary to ON-9S to ON-15S, with biotin at the 3′-end. The
ligation probe DNA was a 9-mer DNA (5′-tcgctattc-3′) with phos-
phate at the 5′-end and digoxigenin at the 3′-end. The template
DNA and the ligation probe DNA were purchased from Japan
Enzymatic digestion study
The sample solutions were prepared by dissolving 0.75 μmol of Bio Service. Reacti-Bind NeutrAvidin-coated polystyrene strip
oligonucleotides in 50 mM Tris-HCl buffer (pH 8.0) containing plates were purchased from Thermo Fisher Scientific (Nunc
10 mM MgCl2. To each sample solution, 0.175 μL Crotalus ada- immobilizer streptavidin F96 white, 436015). The template
manteus venom phosphodiesterase (CAVP) was added and the DNA solution (100 nM) was prepared in hybridization buffer
cleavage reaction was carried out at 37 °C. A portion of each containing 60 mM Na2HPO4 (pH 7.4), 0.9 M NaCl, and 0.24%
reaction mixture was removed at timed intervals and heated to Tween 20. The ligation probe DNA solution (200 nM) was pre-
90 °C for 5 min to deactivate the nuclease. Aliquots of the pared in 1.5 units per well of T4 DNA ligase (TaKaRa) with
timed samples were analyzed by RP-HPLC to evaluate the 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl2, 10 mM DTT and
amount of intact oligonucleotides remaining. The percentage 0.1 mM ATP.
of intact oligonucleotide in each sample was calculated and The washing buffer used throughout the assay contained
plotted against the digestion time to obtain a degradation 25 mM Tris-HCl (pH 7.2), 0.15 M NaCl and 0.1% Tween 20.
curve with time (Fig. S5†).
Anti-digoxigenin-AP antibody (Fab fragments conjugated with
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Organic & Biomolecular Chemistry
alkaline phosphatase) was obtained from Roche Diagnostics. A Grants-in Aid for Scientific Research from the Japanese Minis-
1 : 2000 dilution of the antibody with 1 : 10 super block buffer try of Health, Labor, and Welfare (H23-seisaku tansaku-ippan-
in TBS (Pierce) was used in the assay. The alkaline phospha- 004 and H26-kanjitu-kanen-wakate-008) and the Advanced
tase luminous substrate was prepared in 250 µM CDP-Star Research for Medical Products Mining Programme from the
(Roche) with 100 mM Tris-HCl (pH 7.6) and 100 mM NaCl.
National Institute of Biomedical Innovation. T.Y. thanks the
Assay procedures. Frozen liver tissue was collected in a Grant for Research on Atherosclerosis Update from the Japan
2 mL tube with 1 mL of PBS and a zirconia ball (∅ 5 mm, Irie) Heart Foundation & Astellas/Pfizer. A. Y. thanks the Research
and mechanically homogenized for 2 min at 30 oscillations Fellowship from the Japan Society for the Promotion of
per second by a TissueLyser II apparatus (Qiagen). Total Science (JSPS) for Young Scientists.
protein concentrations were measured using a detergent com-
patible assay kit (Bio-Rad) and adjusted to 8 mg L−1 with PBS.
The assay was performed at the concentration range of 128 pM
to 1000 nM in duplicate. For the standard curve, 7 standard
Notes and references
solutions were prepared. To AON-untreated mice liver homo-
genates were added ON-10S, ON-11S, ON-12S, ON-13S, ON-14S,
and ON-15S solutions to prepare 7 standard samples at a range
of 128 pM to 1000 nM. Next, the template DNA solution (100
µL) and standard solution (10 µL) or liver homogenates (10 µL)
containing ON-10S, ON-11S, ON-12S, ON-13S, ON-14S, and
ON-15S were added to Reacti-Bind Neutr Avidin-coated poly-
styrene strip 96-well plates and incubated at 37 °C for 1 h to
allow the binding of biotin to streptavidin-coated wells and
hybridization. After hybridization, the plate was washed three
times with 200 µL of washing buffer. Then, ligation probe DNA
solution (100 µL) was added, and the plate was incubated at
room temperature (15 °C) for 3 h. The plate was then washed
three times with the washing buffer. Subsequently, 200 µL of a
1 : 2000 dilution of anti-digoxigenin-AP was added, and the
plate was incubated at 37 °C for 1 h. After washing three times
with the washing buffer, the CDP-Star solution was added to
the plate, and finally the luminescence intensity was deter-
mined by using a Centro XS3 luminometer (Berthold) one
second after the addition of CDP-Star. The linear range of
128 pM to 1000 nM in this ELISA system was determined as
r > 0.97.
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Serum chemistry
Serum from the blood collected from the inferior vena cava
upon sacrifice was subjected to serum chemistry. Assay kits
(WAKO) were used to measure serum levels of aspartate
aminotransferase (AST) and alanine aminotransferase (ALT),
which are biomarkers for hepatic toxicity.
Statistics
Statistical comparisons were performed by Dunnett’s multiple
comparison tests. *P < 0.05, **P < 0.01, and ***P < 0.001 were
considered to be statistically significant in all cases. N.S. indi-
cates no statistical significance.
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