Synthesis, gene silencing, and molecular modeling studies of 4′- C -aminomethyl-2′- O -methyl modified small interfering RNAs

Article abstract of DOI:10.1021/jo202666m

The linear syntheses of 4′-C-aminomethyl-2′-O-methyl uridine and cytidine nucleoside phosphoramidites were achieved using glucose as the starting material. The modified RNA building blocks were incorporated into small interfering RNAs (siRNAs) by employing solid phase RNA synthesis. Thermal melting studies showed that the modified siRNA duplexes exhibited slightly lower T_m (～1 °C/modification) compared to the unmodified duplex. Molecular dynamics simulations revealed that the 4′-C-aminomethyl- 2′-O-methyl modified nucleotides adopt South-type conformation in a siRNA duplex, thereby altering the stacking and hydrogen-bonding interactions. These modified siRNAs were also evaluated for their gene silencing efficiency in HeLa cells using a luciferase-based reporter assay. The results indicate that the modifications are well tolerated in various positions of the passenger strand and at the 3′ end of the guide strand but are less tolerated in the seed region of the guide strand. The modified siRNAs exhibited prolonged stability in human serum compared to unmodified siRNA. This work has implications for the use of 4′-C-aminomethyl-2′-O-methyl modified nucleotides to overcome some of the challenges associated with the therapeutic utilities of siRNAs.

Full text of DOI:10.1021/jo202666m

Article
pubs.acs.org/joc
Synthesis, Gene Silencing, and Molecular Modeling Studies of 4′-C-
Aminomethyl-2′-O-methyl Modified Small Interfering RNAs
Kiran R. Gore,† Ganesh N. Nawale,† S. Harikrishna,† Vinita G. Chittoor,† Sushil Kumar Pandey,†
Claudia Hobartner,‡ Swati Patankar,§ and P. I. Pradeepkumar*,†
̈
†Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076, India
‡Research Group Nucleic Acid Chemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen,
̈
Germany
§Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India
S
* Supporting Information
ABSTRACT: The linear syntheses of 4′-C-aminomethyl-2′-O-
methyl uridine and cytidine nucleoside phosphoramidites were
achieved using glucose as the starting material. The modified
RNA building blocks were incorporated into small interfering
RNAs (siRNAs) by employing solid phase RNA synthesis.
Thermal melting studies showed that the modified siRNA
duplexes exhibited slightly lower Tm (∼1 °C/modification)
compared to the unmodified duplex. Molecular dynamics
simulations revealed that the 4′-C-aminomethyl-2′-O-methyl
modified nucleotides adopt South-type conformation in a siRNA duplex, thereby altering the stacking and hydrogen-bonding
interactions. These modified siRNAs were also evaluated for their gene silencing efficiency in HeLa cells using a luciferase-based
reporter assay. The results indicate that the modifications are well tolerated in various positions of the passenger strand and at the
3′ end of the guide strand but are less tolerated in the seed region of the guide strand. The modified siRNAs exhibited prolonged
stability in human serum compared to unmodified siRNA. This work has implications for the use of 4′-C-aminomethyl-2′-O-
methyl modified nucleotides to overcome some of the challenges associated with the therapeutic utilities of siRNAs.
of chemical modifications in the siRNAs.8,14−16 Various types of
INTRODUCTION
RNA interference (RNAi) is a post-transcriptional gene
silencing mechanism, triggered by double-stranded RNAs
called small interfering RNAs (siRNAs), which are 19−21
■
chemical modifications in the sugar [2′-O-methyl,17 2′-fluoro,18
locked nucleic acid (LNA)19 and its analogues,20,21 2′-
methoxyethyl (MOE),17 2′-fluoroarabino (FANA),22 2′-
azido23 etc.], phosphate backbone [phosphorothioate (PS),24
boranophosphate25 etc.], and nucleobase (5-methyl,26 5-
propargyl,26 8-oxoguanosine,27 N2-cyclopentylguanosine,28
etc.) have been used in siRNAs. Many of these modifications
show compatibility to RNAi machinery depending on their
numbers and positions in a siRNA duplex. However, studies
have shown that each modification has its advantages and
drawbacks.8
base pairs long and contain two nucleotide 3′ overhangs.1−3
One strand of the siRNA is the passenger strand with the same
sequence as the target mRNA, while the other strand is the
guide strand, which has the complementary sequence to the
target.4 The RNAi mechanism involves loading of siRNA into
the RNA induced silencing complex (RISC), which is the key
cellular machinery involved in the RNA-mediated gene
silencing process.4,5 Mammalian RISC contains an endonu-
clease enzyme, Argonaute 2 (Ago2), which is responsible for
the removal of the passenger strand and the guide-strand-
mediated site-specific cleavage of mRNA.4,5
Because Ago2 enzyme is highly sensitive to changes in the
grooves and the RNA backbone, many of the currently available
modifications are not suitable for extensive modification of
siRNA duplexes.8,29 Therefore, modifications, which can be
used in minimal number and can impart high therapeutic
potency, are of great value. Toward this end “dual
modifications”, which harbor two modified functionalities on
a single ribose skeleton such as 2′-O-methyl-4′-thioRNA (Me-
SRNA),30 2′-fluoro-4′-thioRNA (F-SRNA),30 and 4′-thio-2′-
fluoro-β-D-arabinonucleic acid (S-FANA)31 units, have been
Because RNAi is a powerful tool, which can be used to
silence expression of any particular gene, it has created great
opportunities to harness the therapeutic potentials of siRNAs.
Several siRNA-based molecules are currently being evaluated in
the clinic.6,7 Despite their significant potential benefits,
numerous challenges are associated with the therapeutic
applications of siRNAs.8 These include their susceptibility
toward nucleases,9 off-target effects,10,11 innate immune
stimulation,12 and inefficient in vivo delivery.13 These challenges
can be addressed to a large extent by the rational incorporation
Received: December 26, 2011
Published: February 28, 2012
© 2012 American Chemical Society
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incorporated into siRNAs. The Me-SRNA showed remarkable
increase in nuclease stability compared to the 2′-O-methyl and
2′-F modified RNAs.30 F-SRNA showed moderate improve-
ment in target binding affinity compared to Me-SRNA. S-
FANA modification has shown compatibility with the RNAi
machinery.31 These interesting findings provide opportunities
to explore new “dual modifications” in siRNAs.
their use in siRNAs has not been found in the literature. We
hypothesize that this modification could impart many desired
therapeutic properties to siRNA. The 4′-C-aminomethyl-2′-O-
methyl modification, being anchored in the sugar, is expected to
be positioned in the minor groove of a siRNA/mRNA duplex.
This is advantageous since the minor groove modifications such
as 2′-O-methyl and 2′-fluoro are known to inhibit immune
stimulation of siRNAs.34 Also, the 2′-O-methyl group is
expected to tune the sugar conformation to C3′-endo, which
is in fact necessary for forming the desired A-type RNA/RNA
helix and also to enhance the target mRNA binding affinity.35
The 2′-O-methyl group at position 2 in the seed region of the
guide strand has been shown to reduce off-target effects
significantly.10,11 The aliphatic primary amine at the 4′-position
can get protonated under physiological pH. This will reduce the
net negative charge of the RNA and thereby enhance its cellular
uptake.36 Moreover, the positively charged amino group could
enhance the nuclease stability of siRNAs.37,38 In this work, we
examine the effect of the 4′-C-aminomethyl-2′-O-methyl
modification on thermal stability, serum stability, and RNAi
activity. In addition, MD stimulations were carried out to
decipher the structural and dynamic features of this
modification in siRNA duplexes.
Here we report the synthesis of 4′-C-aminomethyl-2′-O-
methyl uridine and cytidine (Figure 1) modified siRNAs.
Figure 1. Structures of 4′-C-aminomethyl-2′-O-methyl uridine (1) and
cytidine (2) siRNA modifications reported in this study.
Though 4′-C-aminomethyl-2′-O-methyl thymidine modified
DNAs have been reported by Wengel and co-workers,32,33
the synthesis of the corresponding uridine and cytidine units or
a
Scheme 1. Synthesis of 4′-C-Aminomethyl-2′-O-methyl Uridine Phosphoramidite
a
Reagents and conditions: (i) acetic acid, triflic acid, acetic anhydride, rt, 3 h; (ii) uracil, N,O-bis(trimethylsilyl) acetamide, TMSOTf, CH3CN, 80
°C, overnight; (iii) 4-methoxy benzyl chloride, DBU, CH3CN, rt, 6 h; (iv) NaOMe, MeOH, rt, 3 h; (v) NaH, MeI, THF, rt, 3 h; (vi) (a) Pd(OH)2,
ammonium formate, MeOH, reflux, 6 h, (b) CF3COOEt, Et3N, MeOH, rt, overnight; (vii) CAN, CH3CN/H2O (9:1), rt, 55 h; (viii) (a) PPh3, THF,
H2O, 45 °C, 4 h, (b) CF3COOEt, Et3N, rt, overnight; (ix) 10% Pd/C, EtOH, 70 °C, 16 h; (x) DMT-Cl, pyridine, rt, 24 h; (xi)
NC(CH2)2OP(Cl)N(iPr)2, DIPEA, DCM, rt, 75 min.
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a
Scheme 2. Synthesis of 4′-C-Aminomethyl-2′-O-methyl Cytidine Phosphoramidite
a
Reagents and conditions: (i) acetic anhydride, pyridine, rt, overnight; (ii) TPS-Cl, DMAP, Et3N, DCM, rt, 5 h; (iii) 30% aq NH3, THF, rt,
overnight; (iv) (a) TMS-Cl, pyridine, rt, 1 h, (b) iBu-Cl, rt, 3 h; (v) CN(CH2)2OP(Cl)N(iPr)2, DIPEA, DCM, rt, 40 min.
Table 1. Sequences of siRNA Duplexes Reported in This Study and Representative Tm Values
a
Tm represents melting temperatures for unmodified, 4′-C-aminomethyl-2′-O-methyl (green) and 2′-O-methyl (red) modified siRNA duplexes.
ΔTm represents [Tm (RNAmod) − Tm (RNAunmod)]. The Tm values were determined using 1 μM siRNA in Tm buffer (100 mM NaCl, 10 mM
b
Na2PO4, 0.1 mM EDTA, pH 7.4). All experiments were triplicated, and the Tm values reported are the average of 3 measurements with the estimated
standard deviation. n.d. = not determined
c
temperature for 3 h. Without further purification, using a
modified Vorbruggen reaction,41 the diacetate 4 was subjected
to base coupling using silylated uracil (N,O-bis(trimethylsilyl)
acetamide and uracil) and trimethylsilyl triflate in acetonitrile at
80 °C for overnight to furnish nucleoside 5 in 77% yield. The
β-configuration of the uracil base in 5 was confirmed by 1D
NOE. The H2′ of the sugar showed 8% NOE enhancement
when H6 of the base was irradiated. The N3 of nucleoside 5 was
protected with 4-methoxy benzyl chloride (PMB-Cl) using 1,8-
RESULTS AND DISCUSSION
■
Synthesis of 4′-C-Aminomethyl-2′-O-methyl Modified
Uridine and Cytidine Phosphoramidites and Modified
RNAs. The synthesis of 4′-C-aminomethyl-2′-O-methyl modi-
fied uridine and cytidine blocks started from the known azido
sugar precursor 3 as shown in Scheme 1. The azido sugar 3 was
prepared from D-glucose using reported procedures.39,40,33
Compound 3 was converted to diacetate 4 using a mixture of
acetic acid, acetic anhydride, and triflic acid by stirring at room
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diazabicyclo[5.4.0]undec-7-ene (DBU) as a base in acetonitrile
at room temperature for 6 h to furnish compound 6 in 89%
yield.42 The 2′-acetate group of nucleoside 6 was hydrolyzed
using 1 M sodium methoxide in methanol at room temperature
for 3 h to yield 2′-hydroxy nucleoside 7. Crude 7 was directly
methylated by using methyl iodide (MeI) and sodium hydride
(NaH) in tetrahydrofuran (THF) at room temperature for 3 h
to obtain 2′-O-methyl nucleoside 8 in 88% yield.41 The
debenzylation and azide reduction of 8 were attempted using
ammonium formate and 20% Pd(OH)2 in methanol at reflux
condition for 6 h, followed by protection of the 4′-CH2NH2
group using ethyl trifluoro acetate (CF3COOEt) and triethyl-
amine (Et3N) in MeOH at room temperature overnight.41,43
However, during these processes, the C5−C6 double bond of
the uridine of 8 was reduced to yield dihydrouridine nucleoside
9 bearing a PMB group in 71% yield. Similar to our
observation, the formation of undesired dihydrouridine product
has been reported during the debenzylation of LNA building
integrity of the RNAs was confirmed by negative mode ESI-
MS analysis (Table S10, Supporting Information).
Thermal Melting Studies of siRNA Duplexes. To
explore the effect of chemical modification on siRNA duplex
stability, UV-melting studies were performed in phosphate
buffer (pH 7.4) containing 100 mM NaCl (Figure S1,
Supporting Information). The Tm values are reported in
Table 1. Incorporation of 2′-O-methyl modification in an
siRNA duplex enhanced the Tm by 0.5 °C/modification, which
is in agreement with the previous studies.50 In contrast,
depending upon the position in the siRNA, the presence of a
single 4′-C-aminomethyl-2′-O-methyl uridine/cytidine or two
uridines decreased the Tm by 0.5−1.3 °C/modification. A
similar decrease in Tm (∼2.6 °C/modification) was observed
when 4′-C-aminomethyl-2′-O-methyl thymidine modifications
were incorporated into a 2′-O-methyl RNA.33 Matsuda and co-
workers also showed that various 4′-C-aminoalkyl deoxythymi-
dines cause a slight decrease in melting temperatures of DNA-
RNA duplexes (∼1 °C per modification).38 The lower Tm
values indicate that the presence of the 4′-C-aminomethyl
group may have affected the sugar conformation and the
hydration at the minor groove, thereby creating disturbances in
the stacking and hydrogen bonding interactions. We have
addressed these aspects in detail in the molecular modeling
section. It should be noted that the slight decrease in thermal
stability observed for 4′-C-aminomethyl-2′-O-methyl uridine
and cytidine modified siRNAs was not detrimental for RNAi
activity (see the following section for details).
RNA Interference Studies in Cellular Systems. The
modified siRNAs were tested for their RNAi efficiency using
transfection studies of a luciferase reporter system in HeLa
cells. Since the modified siRNAs targeted the Renilla luciferase
gene, the psiCHECK2 plasmid containing Renilla (target) and
Firefly (control) luciferase genes was used as the reporter
system. Lipofectamine-2000 was used as transfection agent for
the plasmid and siRNA delivery.22 The HeLa cells were
transfected with siRNA (33 nM), and expression of both
luciferase genes was analyzed after 48 h using the dual luciferase
reporter assay (DLRA). The scrambled (SC) siRNA was used
as negative control in the experiments. Only psiCHECK2
plasmid was used in the positive control (PC) experiments.
Gene silencing efficiency was measured by dividing Renilla
luciferase light units by Firefly luciferase light units and
normalized with respect to the positive control. Efficient gene
silencing is indicated by low normalized ratios of Renilla/Firefly
luciferase activity. The results are summarized in Figure 2.
The results showed that the modifications are well tolerated
at various positions in the passenger strand of siRNA. All
passenger strand modified siRNA duplexes such as 6R-2R, 9R-
2R, and 16R-2R showed almost no loss of RNAi activity when
compared to the unmodified duplex, 3R-2R. The modification
in the 3′-overhang of passenger strand as in 12R-2R was also
well tolerated. Therefore, it is clear that the passenger strand
modifications do not disturb the binding or function of Ago2
protein in the RISC. The effect of modifications in the guide
strand was found to be position-dependent. The cytidine
modification toward the 3′-end of the guide strand as seen in
3R-22R is well tolerated. The modification at position 3 of the
seed region of the guide strand as in 3R-7R and 6R-7R showed
decrease in RNAi activity by 35% and 45% respectively. It
should be noted that the 2′-O-methyl modified siRNA duplex,
3R-29R, in which similar positions are modified as 3R-7R,
retained the RNAi activity. This shows that the decrease in
44
blocks using 20% Pd(OH)2. To solve this problem, we
employed an alternative strategy for deprotection involving
multiple steps. First, the deprotection of the PMB group of
compound 8 was performed using ceric ammonium nitrate
(CAN) in aqueous acetonitrile (9:1) at room temperature for
55 h to furnish compound 10 in 76% yield.43 Reduction of the
azide group of compound 10 was carried out with
triphenylphosphine (PPh3) in THF at 45 °C for 4 h to furnish
the crude amine, which was subsequently protected by using
CF3COOEt at room temperature for overnight to afford the
protected nucleoside 11 in quantitative yield.45 Debenzylation
of compound 11 was carried out with 10% Pd/C in EtOH at 70
°C for 16 h to give the deprotected nucleoside 12 in 71% yield.
The 5′-OH of nucleoside 12 was protected using 4,4′-
dimethoxytrityl chloride (DMT-Cl) in pyridine at room
temperature for 24 h to furnish compound 13 in 81% yield.43
Nucleoside 13 was phosphitylated using 2-cyanoethyl- N,N-
diisopropylchloro phosphoramidite (CEP-Cl) and N,N-diiso-
propylethylamine (DIPEA) in dichloromethane (DCM) at
room temperature for 75 min to furnish the phosphoramidite
14 in 70% yield.46
The synthesis of cytidine phosphoramidite 19 was started
from compound 13 as outlined in Scheme 2. Acetylation of
DMT uridine nucleoside 13 was achieved using acetic
anhydride in pyridine to afford acetylated nucleoside 15. The
crude 15 was converted to 2,4,6-triisopropylbenzenesulphonyl
(TPS) derivative 16 using TPS-Cl, 4-dimethylaminopyridine
(DMAP), and Et3N in DCM at room temperature. Without
further purification, the TPS derivative 16 was treated with 30%
aqueous NH3 in THF for 12 h at room temperature to furnish
cytidine nucleoside 17 in 63% yield (after three steps).47 The
exocyclic amino group of 17 was protected using isobutyryl
chloride (iBu-Cl) in pyridine at room temperature for 3 h
employing transient protection of the hydroxyl group by
trimethylsilyl (TMS-Cl) to furnish compound 18 in 67%
yield.43 The cytidine nucleoside 18 was phosphitylated using
CEP-Cl and DIPEA in DCM at room temperature for 40 min
to furnish the phosphoramidite 19 in 51% yield.
The modified phosphoramidites 14 and 19 were successfully
incorporated into siRNAs (Table 1), designed to target the
Renilla luciferase gene using standard solid phase RNA
synthesis. The chemical modifications are judiciously incorpo-
rated at various positions in the passenger strand and guide
strands. The RNAs were deprotected using standard con-
ditions48,49 and purified by 20% denaturing PAGE. The
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Figure 3. PAGE denoting stability of siRNA duplexes (1.5 μM) in
human serum. 32P-labeled siRNA guide strands 2R, 22R, and 7R
annealed with cold complementary passenger strand 3R and incubated
in 10% human serum for various time points (t = 0, 0.25, 0.5, 1, 2, 4, 8,
22, and 44 h). The intensity of the full-length siRNA bands decreases
with increase in incubation time. The arrow represents the full length
siRNA guide strand.
Figure 2. RNAi activity of unmodified and modified siRNAs after 48 h
of transfection. HeLa cells were transfected with psiCHECK2 plasmid
and siRNAs. Normalized luciferase activity was measured by dividing
Renilla luciferase light units by Firefly luciferase light units and
normalized with respect to the positive control (PC, no siRNA). SC
denotes scrambled siRNA. Statistical analyses between individual
groups were performed by a Student t test; a P value of <0.05 was
considered statistically significant. All error bars represent standard
error of the mean (SEM) with n = 4.
increase the serum stability considerably. However, we expected
a higher enhancement of the serum stability, since protonated
2′- and 4′-aminoalkyl groups were reported to generate
unfavorable electrostatic interactions with the cationic domain
of nuclease enzymes.38,52 The MD simulation studies (see the
following section for details) have shown that the 4′-C-
aminomethyl group in the sugar is flexible and does move away
from phosphate group without having any significant electro-
static interaction. This may be the reason for the moderate
serum stability observed for the modified siRNAs. Keeping in
view the tolerance of 4′-C-aminomethyl-2′-O-methyl modifica-
tions at the 3′-ends of siRNA by the RNAi machinery, our
results indicate that introduction of 1−2 modifications at the 3′-
ends will be a reliable strategy to enhance serum stability.
Molecular Modeling Studies. The goal of this MD
simulation study was to explore the real time dynamics of the
solvated siRNA duplexes and to elucidate atomic-resolution
details of the structure. Also, the effect of 4′-C-aminomethyl-2′-
O-methyl modified nucleotides on the thermal stability and
nuclease stability of siRNA duplexes could be addressed using
MD simulation. The two modified siRNA duplexes 3R-7R and
6R-7R were chosen for MD studies (Table 1). The siRNA
duplex 3R-7R contains modified uridine at positions 3 and 21
of the guide strand. Here, the modifications lie in the seed
region and the 3′ overhang of siRNA duplex. The siRNA duplex
6R-7R contains four modifications, two at the 5′ end of the
passenger strand, one at the seed region, and the other at the 3′
overhang of the guide strand. The unmodified siRNA duplex
3R-2R was used to compare the structural variability in the
modified duplex after 20 ns of MD simulation.
RNAi activity of the seed region modified duplexes is due to the
presence of the 4′-C-aminomethyl group, which perhaps
hinders a key nucleic acid−protein interaction of the RNAi
machinery. This would be expected as the seed region is very
important in recognizing the target mRNA sequence.10 This
position has also been shown to play an important role in the
formation of the RISC complex, since the MID domain of Ago2
strongly interacts with the 5′-end of the guide strand.8,51
Serum Stability Studies. Unmodified siRNAs are highly
unstable in serum because of their susceptibility to rapid
degradation by nuclease enzymes. Therefore, chemical
modifications can increase the half-life of siRNAs and thereby
prolong RNAi activity in vivo.8 We assessed the effect of
chemical modifications on the stability in human serum of two
modified siRNA duplexes: 3R-22R, which shows in vivo RNAi
activity similar to that of the unmodified duplex 3R-2R, and
3R-7R, which shows reduced in vivo RNAi activity compared to
that of 3R-2R. Serum stability of these modified siRNAs was
compared with that of the unmodified siRNA. The 32P-labeled
modified guide strands 2R, 22R, and 7R were annealed with
cold complementary unmodified passenger strand 3R, and
incubated in 10% human serum. Their stability toward serum
derived nuclease digestion at various incubation time points
was analyzed by denaturing PAGE (Figure 3). The unmodified
siRNA duplex was 100% degraded by serum nucleases within 1
h of incubation time. The modified siRNA duplexes were found
to be stable for an extended period of time compared to the
unmodified siRNA.
The geometry of 4′-C-aminomethyl-2′-O-methyl nucleotide
was optimized (HF/6-31G*), using Gaussian03,53 which
showed the C3′-endo sugar conformer (Figure S2, Supporting
Information). Using the antechamber RESP fitting procedure,
the charges for the modified uridine atoms were obtained. The
new force field parameters and RESP charges derived for
The 3R-22R siRNA with just one cytidine modification
toward the 3′-end of the guide strand was intact for 2 h in
serum. The 3R-7R having modifications in position 3 of the
seed region and in the overhang was found to be intact up to 8
h. These results clearly show that overhang modifications
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modified nucleotides were incorporated into the siRNA duplex
at respective positions and solvated using water. The solvated
siRNA system was then subjected to equilibration, followed by
unrestrained MD simulation for 20 ns using AMBER 10.54 All
three duplexes were started with A-form RNA geometry and
retained this general form over 20 ns of MD simulation (Figure
S3, Supporting Information).
The stability of RNA duplex was monitored by calculating
the root-mean-square deviation (rmsd, only heavy atoms) of
the conformation evolved during each picosecond of MD
simulation (Figure S4, Supporting Information). The rmsd
graph over 20 ns trajectory showed that modified duplexes
form more flexible structures when compared to the
unmodified siRNA duplex. All three duplexes were stabilized
and the rmsd converged after 4−5 ns. The positional rmsd
calculations around the modified positions (Table S1,
Supporting Information) suggest that the modified nucleotides
in the helical region of the passenger and the guide strand and
also at the overhang region of guide strand are highly flexible.
The root-mean-square fluctuation (rmsf) of backbone atoms
was also calculated for each duplex (Figure S5, Supporting
Information). The rmsf values imply that RNA backbones of
modified duplexes were more flexible (rmsf = 7.0 Å) than the
unmodified siRNA duplex (rmsf = 5.2 Å).
In order to probe the influence of 4′-C-aminomethyl-2′-O-
methyl modification on the sugar puckering, the percentage of
C3′-endo sugar conformation was calculated from 20 ns MD
trajectories (Figure S6, Supporting Information). Previous
studies have shown that natural 2′-OH, and 2′-O-methyl
modification in the ribose sugar drives the sugar conformation
strongly to North-type C3′-endo pucker in an RNA duplex.55−57
This behavior was also expected for the 4′-C-aminomethyl-2′-O-
methyl modified nucleotides. In contrast, strong South-type
C2′-endo conformation was significantly visible at the modified
nucleotide positions of 3R-7R and 6R-7R (Figure 4 and Figure
1.5 and 1.7 °C, respectively. NMR studies at the nucleoside
level, DFT calculations performed in both gas phase and in
solution for 2′-amino substituted residues,59,60,64 and MD
simulations for the 2′-S-methyl modified duplex65 all suggest
that the S type C2′-endo puckering is energetically favored by
these modifications. These studies identified that a combination
of steric factors, reduced electronegativity, weaker gauche effect,
and reduced hydration are responsible for the adaptation of
C2′-endo conformation, which causes the duplex destabiliza-
tion. In contrast, X-ray structural data of 2′-amino,66 2′-amido,66
and 2′-S-methyl67 modified duplexes reveal that modified
nucleotides switch to C3′-endo conformation in the crystal
state. It is interesting to note that recent Selective 2′-Hydroxyl
acylation Analyzed by Primer Extension (SHAPE) experiments
underscore the fact that local conformational changes of the
sugar moieties in an RNA duplex can be induced by the buffer
conditions used for crystallization.66,68 Therefore, the con-
formational flexibility and the sugar pucker preference of
modified nucleotides responsible for changes in the thermody-
namic stability of RNA duplexes may not be evident from the
crystal structures.
The conformation of phosphodiester backbone angles
(α−ζ), which are closely related to sugar puckering, was
found to be gauche− trans (g− t) in the 4′-C-aminomethyl-2′-O-
methyl modified positions of the siRNA duplex. The γ angle
(C4′−C5′) fluctuated because of the flexible movement of 4′-C-
aminomethyl modification. As a result, the expected electro-
static interaction between phosphate group and the amino-
methyl group was not observed. The alternative sugar and
phosphate conformations in RNA duplex are known to increase
the minor groove widths,69 and this was in fact observed in the
modified siRNA duplexes (Figure S8, Supporting Information).
To characterize the general structure and dynamic features of
the duplexes, we calculated the interstrand and intrastrand
phosphate distances. The interstrand phosphate distances of
unmodified and modified duplexes depict that all duplexes are
in its secure girth (Figure S8, Supporting Information). At the
modified nucleotide position, the interstrand phosphate
distance was elongated up to 23 Å. The nucleotides in the
unmodified siRNA duplex have interstrand phosphate distances
of only 18 Å. Also, the intrastrand phosphate distances of
modified nucleotides extend up to 7.0 Å compared to the 5.9 Å
distances of unmodified nucleotides in siRNA (Figures S9−
S11, Supporting Information). This longer distance means that
the 4′-C-aminomethyl-2′-O-methyl modified nucleotides deviate
from the typical layout of A-form helix, thereby affecting the
groove geometry.
Figure 4. MD snapshots at the modified positions showing the base
pairing nucleotide (U-A) and the nature of sugar conformation. (A)
Nucleotides of the unmodified siRNA in C3′-endo sugar conforma-
tion. (B) 4′-C-Aminomethyl-2′-O-methyl (green) nucleotide of the
modified siRNA in C2′-endo sugar conformation. Black dotted lines
represent the hydrogen bond interaction between the bases.
Since aqueous solvent performs a major role in the nucleic
acid structure stability and dynamics,70 the first hydration shell
(<3.0 Å) of the duplex groove and phosphate backbone were
computed (Table S2, Supporting Information). The higher
thermodynamic stability of RNA over DNA is often ascribed to
the presence of a 2′-OH group, which can form a stable solute−
solvent interaction and contribute to the stabilization of the
hydration shell.71 RNA duplexes containing 2′-O-methyl
modified nucleotides are more stable than the unmodified
nucleotides because of long-lived hydration patterns observed
in their rigid grooves.72 Our MD simulation studies of modified
siRNA duplexes showed that 4′-C-aminomethyl-2′-O-methy-
lated nucleotides failed to create optimal water binding pockets,
which could unfavorably contribute to the thermodynamics of
hydration. Minor groove hydration in the unmodified siRNA
was found to be higher (∼17 water molecules) than in the
S6, Supporting Information). The amplitude of the pseudo
rotational angle (tm),58 which represents the nature of four
dihedral angles in sugar, showed that all of the unmodified
nucleotides in the modified siRNA duplexes retained the native
C3′-endo sugar conformation (Figure S6, Supporting Informa-
tion). The presence of C2′-endo puckered sugars in a
predominately C3′-endo dominated A-type helix is likely to
be responsible for the slight loss in the thermodynamic stability
(∼1 °C/modification) observed for the modified siRNA
duplexes. It is reported in the literature that some of the 2′-
modifications such as 2′-amino59 and 2′-amido60 decrease the
thermodynamic stability of RNA/RNA duplex by around 4.3
and 7.3 °C per modification, respectively. Also, the presence of
a single 2′-S-methyl,61,62 and 2′-alkyl63 modification in a DNA/
RNA duplex decreases the thermodynamic stability by around
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modified siRNA duplexes (∼10 water molecules) (Table S2,
Supporting Information). The flexibility of helices and long
inter- and intrastrand phosphate distances led to unstable
minor groove dimensions in the modified siRNA duplex and
thus failed to hold the water molecule compactly (Figure S12,
Supporting Information). The hydration numbers for backbone
atoms and major grooves were also calculated, which revealed
that backbone atoms and major grooves in the modified and
unmodified duplexes were almost equally hydrated.
The helical deformation and stacking parameters were
calculated for all of the duplexes to delineate the effects of
modification on helical and stacking stability of RNA. The
overall results of these parameters indicate that the
modification affects the stacking and helical stability of siRNA
(Figure 5). The values of buckle, open angle, stagger, propeller,
position in 3R-2R (Figure S13, Supporting Information). The
capability of holding the base in proper orientation, such that it
can form stable hydrogen bond and stacking interaction with
nearby and pairing base, can be explained by the χ angle.74 The
χ angle for typical A-type RNA is −160° and for DNA is
−120°.75 Here, for the 4′-C-aminomethyl-2′-O-methyl modified
nucleotides the χ angle was found to be −120° (Figure S13,
Supporting Information). This shows that 4′-C-aminomethyl-2′-
O-methyl modified nucleotides deviate from the original RNA
type to DNA type, which reflects a scuffle in hydrogen bond
and stacking interactions. To address this in detail, the
occupancy of two hydrogen bonds between the modified
nucleotide and the pairing base was determined. The hydrogen
bond occupancy was lacking up to 50% at the modified regions
(Figure 5 and Table S8, Supporting Information), which was
obviously due to the flexibility of backbone atoms and deviated
χ angle.
To obtain deeper insights into the energetics of the hydrogen
bond and stacking interactions at the modified nucleotides in
the siRNA, the single point energy at DFT (MP2(full)/6-
311G*) level was computed (Table S9, Supporting Informa-
tion).76 The hydrogen bond energy (ΔEHB) at the modified
position and interstrand stacking energy (ΔEinter) at the hexa-
nucleotide base pairs in which the modified nucleotides are in
the middle were calculated (Figure S14, Supporting Informa-
tion). The intrastrand stacking energy (ΔEintra) was also
calculated for trinucleotides in which the modified nucleotide is
positioned in the middle. The ΔEHB values at the modified
nucleotides were higher than the bonding energy between two
unmodified nucleotides (Table S9, Supporting Information).
The same trends were observed in the case of ΔEintra. The
ΔEinter was almost similar in guide strand modified, 3R-7R, and
unmodified, 3R-2R, siRNAs. However, the siRNA duplex 6R-
7R containing modifications in the passenger and guide strand
had higher ΔEinter (Table S9, Supporting Information).
Taken together, MD analysis (20 ns) shows how the 4′-C-
aminomethyl-2′-O-methyl modified nucleotides in the siRNA
duplexes induce changes in the sugar puckering, flexibility in
groove dimension, and disturbances in hydrogen bonding and
base stacking when compared to the unmodified siRNA. This
results in the slight decrease in the thermodynamic stability of
the modified siRNA duplexes that was observed in the UV-
melting studies.
Figure 5. Final MD snapshot of the duplexes at the unmodified and
modified region (helical and top view) captured after 20 ns. (A)
Unmodified siRNA duplex, 3R-2R shows well stacked architecture of
nucleotides. (B) 4′-C-Aminomethyl-2′-O-methyl modification (green)
in siRNA duplex, 3R-7R, shows disturbance in stacking interaction in
the helical region. (C) Hydrogen bonding and stacking interaction
between the two successive nucleotide pairs (A-U, C-G) of
unmodified siRNA duplex, 3R-2R. (D) Disturbed stacking and
hydrogen bond interactions at the modified nucleotide (green) of
siRNA duplex, 3R-7R. Black dotted lines in panels C and D represent
the hydrogen bond interaction between the bases.
and rise at the unmodified regions of the helices in 3R-7R and
in the unmodified siRNA 3R-2R were found to be similar
(Table S3 and S4, Supporting Information). On the other hand,
siRNA duplex 6R-7R showed higher values (up to 30 Å) of
buckle and open angle parameters at modified nucleotide
positions (Table S5, Supporting Information). Moreover,
stretch and shear parameters were found to be higher for the
modified siRNAs than in the unmodified duplex (Tables S4 and
S5, Supporting Information). These observations indicate that
the minor and major groove widths were fluctuating during
simulation. Of the six intrastrand stacking parameters, there was
less deviation in roll and rise parameters of modified siRNA in
comparison to the unmodified siRNA. However, in the
modified duplexes, twist and tilt angles were highly deviated
(Table S6 and S7, Supporting Information); this reflects slight
unwinding of the helix.73
CONCLUSIONS AND OUTLOOK
■
Syntheses of 4′-C-aminomethyl-2′-O-methyl uridine and
cytidine nucleoside phosphoramidites and the corresponding
siRNAs have been achieved. Thermal melting studies of
modified siRNA showed a ∼1 °C reduction in Tm per
modification when compared to the unmodified duplexes. This
modest destabilizing modification can perhaps be utilized to
increase the thermodynamic asymmetry in siRNA duplexes,
thereby enhancing their target specificity and potency.20 Gene
silencing studies in cellular systems showed that the 4′-C-
aminomethyl-2′-O-methyl modification is compatible with the
RNAi machinery, but the tolerance is position-dependent. The
modifications are well tolerated at different positions in the
passenger strand and at the 3′-end of the guide strand but less
tolerated in the seed region of the guide strand. The decreased
activity suggests that the 4′-C-aminomethyl group in the seed
region is not optimally compatible with formation of an
efficient RISC complex, possibly because of sterically
unfavorable interactions with Ago2 protein. More structure−
Frequency of chi (χ) angle at the junction of sugar and base
was determined for modified nucleotides in 3R-7R and 6R-7R
and compared with the unmodified nucleotides at respective
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by employing standard phosphoramidite chemistry. Mass spectra of
oligonucleotides were obtained in negative ESI mode.
activity studies replacing every nucleotide in the guide strands
of siRNA are required to decipher complete positional
tolerance of the modification.77
1-[2′-O-Acetyl-3′,5′-di-O-benzyl-4′-C-azidomethyl-β-D-ribo-
furanosyl]-uracil (5). Acetic anhydride (7.48 mL, 79.3 mmol) and
acetic acid (40 mL) were added to compound 3 (2.6 g, 6.35 mmol).
The mixture was cooled to 4 °C, and triflic acid (0.03 mL, 0.32 mmol)
was added in dropwise. After stirring for 3 h at room temperature, the
reaction mixture was quenched with cold saturated NaHCO3 (60 mL)
and extracted with ethyl acetate (3 × 150 mL). The organic layer was
separated, dried over Na2SO4, and evaporated to give crude compound
4 (2 g, 4.7 mmol), which was co-evaporated with toluene (2 × 70 mL)
and dried under high vacuum. The crude product 4 was dissolved in
CH3CN (47 mL). To this were added uracil (631 mg, 5.64 mmol) and
N,O-bis(trimethylsilyl) acetamide (3.2 mL, 13.16 mmol), and the
mixture was heated at 80 °C for 45 min until the suspension became a
clear solution. This solution was cooled to 0 °C, and TMSOTf (1.1
mL, 6.11 mmol) was added in dropwise, warmed to 80 °C, and stirred
overnight. The reaction was quenched with saturated NaHCO3
solution (80 mL) and extracted with ethyl acetate (3 × 100 mL).
The organic layer dried over Na2SO4, evaporated, and then purified by
column chromatography (30−40% ethyl acetate in hexane) to yield
compound 5 as white solid (1.88 g, 77%); Rf = 0.72 (70% ethyl acetate
The presence of just two 4′-C-aminomethyl-2′-O methyl
modifications in the siRNA guide strand resulted in higher
serum stability compared to unmodified siRNA. This provides
an opportunity to design siRNA containing a minimal number
of modifications without sacrificing RNAi activity as well as
nuclease stability. Minor groove base modifications are known
to inhibit the off-target effects and immune stimulation.78 Since
the 4′-C-aminomethyl-2′-O-methyl modification projects into
the minor groove, our modified siRNAs will be evaluated
further for off-target and immune-stimulation effects. Since the
effect of 4′-modifications on siRNA activity and off-target
effects are rarely found in the literature, this modification
provides an opportunity to unravel the structural and functional
requirements of RNAi at the 4′-position. The 4′-C-aminomethyl
groups can also be utilized to conjugate suitable molecules to
siRNAs to enhance their in vivo delivery.79,80
MD simulations show that the overall structure of modified
siRNA retains the A-type RNA helix with higher backbone
flexibility and slight unwinding of the helices at the modified
positions. The modification tunes the sugar puckering to C2′-
endo South-type conformation, and the 4′-C-aminomethyl
group has been found to have no significant interaction with
the phosphate backbone. Modified siRNAs lose hydration in
the minor groove; however, major groove and backbone
hydration were retained when compared to the unmodified
siRNA. We observed that hydrogen bonding and stacking
energies were lower at the modified nucleotide positions in the
siRNA. These local structure alterations result in lower
thermodynamic stability of modified siRNA when compared
to unmodified duplex, and indeed this was observed in our
experimental studies. However, more structural studies are
required to confirm the local conformational perturbations
observed in the MD simulations. Our current modeling studies
are directed toward deciphering the effect of this modification
in the seed region of siRNA on RNAi by employing the siRNA-
argonaute complexes. Overall, 4′-C-aminomethyl-2′-O-methyl
modification is a potent RNAi-compatible chemical modifica-
tion, which offers enhanced serum stability and therefore can
further be evaluated for siRNA therapeutics.
1
in pet ether); mp 123−124 °C; H NMR (400 MHz, CDCl3) δ 8.73
(bs, 1 H), 7.64 (d, J = 7.93 Hz, 1 H), 7.42−7.22 (m, 11 H), 6.18 (d, J
= 4.8 Hz, 1 H) 5.40 (t, J = 5.18 Hz, 1 H), 5.34 (d, J = 8 Hz, 1 H), 4.63
(d, J = 11 Hz, 1 H), 4.49−4.41 (m, 3 H), 4.38 (d, J = 6 Hz, 1H), 3.77,
3.48 (AXq, J = 10.3 Hz, 2 H), 3.66, 3.36 (AXq, J = 13.4 Hz, 2 H), 2.12
(s, 3 H). 13C NMR (100 MHz, CDCl3) δ 170.0, 163.5, 150.5, 140.4,
137.1, 136.9, 128.8, 128.7, 128.5, 128.4, 128.3, 128.2, 102.8, 87.6, 87.5,
75.1, 74.6, 73.9, 71.2, 52.9, 20.7; HRMS (ESI) calcd for C26H28N5O7
[M + H]+ 522.1989, found [M + H]+ 522.1997 (Δm +0.0008, error
+1.6 ppm).
1-[2′-O-Acetyl-3′,5′-di-O-benzyl-4′-C-azidomethyl-β-D-ribo-
furanosyl]-3-(4-methoxybenzyl)-uracil (6). To a mixture of
compound 5 (3.65 g, 7 mmol) and 4-methoxybenzyl chloride (1.7
mL, 12.6 mmol) in CH3CN (50 mL) was added DBU (1.95 mL, 14.03
mmol) at 0 °C, and the mixture was stirred at room temperature for 6
h. After cooling to 0 °C, the mixture was diluted with 5% KHSO4
solution (10 mL). The organic phase was separated, and the aqueous
phase was extracted with ethyl acetate (3 × 80 mL). The combined
organic layer was washed with brine (100 mL), dried over anhydrous
Na2SO4, and evaporated under reduced pressure to give a light yellow
residue. Residue was purified by column chromatography (5−20%
ethyl acetate in pet ether) to yield compound 6 as colorless oily liquid
1
(4.0 g, 89%); Rf = 0.68 (40% ethyl acetate in pet ether); H NMR
(400 MHz, CDCl3) δ 7.6 (d, J = 8.4 Hz, 1 H), 7.42−7.21 (m, 13 H),
6.82−6.79 (m, 2 H), 6.18 (d, J = 4.4 Hz, 1 H), 5.40−5.37 (m, 2 H),
5.04, 4.94 (ABq, J = 13.2 Hz, 2 H), 4.62, 4.43 (AXq, J = 11.6 Hz, 2 H),
4.44, 4.39 (ABq, J = 11.2 Hz, 2 H), 4.37 (d, J = 5.8 Hz, 1H), 3.75, 3.46
(AXq, J = 10.2 Hz, 2 H), 3.76 (s, 3 H), 3.66, 3.35 (AXq, J = 13.3 Hz, 2
H), 2.10 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 169.8, 162.5, 159.2,
151.1, 137.9, 137.2, 137.0, 130.7, 129.0, 128.8, 128.7, 128.6, 128.5,
128.4, 128.2, 128.2, 114.1, 113.8, 102.3, 88.3, 87.4, 77.1, 75.1, 74.5,
73.9, 71.1, 65.1, 55.4, 55.3, 52.9, 43.7, 20.8; HRMS (ESI) calcd for
C34H36N5O8 [M + H]+ 642.2564, found [M + H]+ 642.2584 (Δm
+0.002, error +3.2 ppm).
1-[2′-O-Methyl-3′,5′-di-O-benzyl-4′-C-azidomethyl-β-D-ribo-
furanosyl]-3-(4-methoxybenzyl)-uracil (8). Compound 6 (200
mg, 0.32 mmol) was dissolved in dry methanol (2 mL), to which 1 N
NaOMe (0.57 mL) was added and stirred at room temperature for 3 h.
The solvent was partially evaporated under reduced pressure and
extracted with ethyl acetate (3 × 40 mL). The combined organic phase
was washed with brine (40 mL), dried over anhydrous Na2SO4, and
concentrated. The crude compound 7 obtained was co-evaporated
with toluene (2 × 40 mL) and dried under high vacuum. Then
compound 7 (146 mg, 0.24 mmol) was dissolved in dry THF (2 mL),
and NaH (12 mg, 0.30 mmol) was added and stirred at 0 °C for 5 min.
Then methyl iodide (0.02 mL, 0.35 mmol) was added in dropwise
under nitrogen atmosphere and stirred at room temperature for 3 h.
Saturated NaHCO3 solution (50 mL) was added, the organic phase
EXPERIMENTAL SECTION
■
General. All chemicals and dry solvents (THF, MeOH, and DMF)
were obtained from commercial sources and used without any further
purification. Acetonitrile, DCM, Et3N, DIPEA, and pyridine were dried
using calcium hydride. Toluene was dried using calcium chloride. Thin
layer chromatography (TLC) was performed on silica gel plates
precoated with fluorescent indicator with visualization by UV light or
by dipping into a solution of 5% (v/v) conc H2SO4 in ethanol and
heating. Silica gel (100−200 mesh) was used for column
chromatography. 1H NMR (400 or 300 MHz), 13C NMR (100
MHz), 31P NMR (162 MHz), and 19F NMR (376 or 282 MHz) were
recorded on a 400 or 300 MHz instrument. The chemical shifts in
parts per million (ppm) are reported downfield from TMS (0 ppm) or
1
methanol-d4 (3.31 ppm) for H NMR spectra; CDCl3 (77.2 ppm) or
methanol-d4 (49.1 ppm) for 13C NMR spectra. Multiplicities of H
1
NMR spin couplings are reported as s for singlet, bs for broad singlet,
d for doublet, t for triplet, q for quartet, sep for septet, dd for doublet
of doublet, ABq for AB quartet, AXq for AX quartet, or m for multiplet
and overlapping spin systems. Values for apparent coupling constants
(J) are reported in Hz. High resolution mass spectra (HRMS) were
obtained in positive ion electrospray ionization (ESI) mode. RNA
sequences were synthesized using automated solid phase synthesizer
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was separated, and the aqueous phase was extracted with DCM (3 ×
50 mL). The combined organic layer was washed with brine (40 mL),
dried over anhydrous Na2SO4, and concentrated under reduced
pressure. Crude residue was purified by column chromatography (12−
14% ethyl acetate in pet ether) to afford 8 as yellow oily liquid (131
mg, 88%, after two steps); Rf = 0.83 (40% ethyl acetate in pet ether);
1H NMR(400 MHz, CDCl3) δ 7.87 (d, J = 7.6 Hz, 1 H), 7.43−7.18
gummy colorless compound 11 (1.268 g, 100%); Rf = 0.58, (40% ethyl
acetate in pet ether); 1H NMR (300 MHz, CD3OD) δ 7.93 (d, J = 8.1
Hz, 1 H), 7.38−7.21 (m, 11 H), 6.04 (d, J = 0.9 Hz, 1 H), 5.11 (d, J =
8.1 Hz, 1 H), 4.71, 4.49 (AXq, J = 11.7 Hz, 2 H), 4.44, 4.38 (ABq, J =
10.9 Hz, 2 H), 4.42 (d, J = 5.9 Hz, 1 H), 3.98 (dd, J = 5.7 Hz, 0.9 Hz,
1H), 3.83, 3.61 (ABq, J = 14.5 Hz, 2 H), 3.68, 3.56 (ABq, J = 10.7 Hz,
2 H), 3.55 (s, 3 H); 13C NMR (75.4 MHz, CD3OD) δ 166.1, 159.44,
151.7, 142.4, 138.9, 138.8, 133.8, 133.8, 133.4, 133.2, 133.1, 132.0,
130.1, 129.9, 129.6, 129.5, 129.3, 129.2, 129.2, 119.5, 115.7, 102.1,
90.6, 88.3, 84.3, 77.0, 74.6, 73.8, 71.1, 59.5, 42.7; 19F NMR (282 MHz,
CD3OD) δ −76.2 (s); HRMS (ESI) calcd for C27H29F3N3O7 [M +
H]+ 564.1958, found [M + H]+ 564.1973 (Δm +0.0016, error +2.8
ppm).
(m, 13 H), 6.81 (d, J = 8.2 Hz, 2 H), 6.05 (d, J = 2 Hz, 1 H), 5.21 (d, J
= 8.4 Hz, 1 H), 5.04, 4.94 (ABq, J = 13.6 Hz, 2 H), 4.70, 4.44 (AXq, J
= 11.8 Hz, 2 H), 4.46, 4.40 (ABq, J = 10.9 Hz, 2 H), 4.26 (d, J = 5.6
Hz, 1 H), 3.95, 3.29 (AXq, J = 13.6 Hz, 2 H), 3.93, 3.52 (AXq, J = 10.4
Hz, 2H), 3.79−3.73 (m, 4 H), 3.57 (s, 3 H); 13C NMR (100 MHz,
CDCl3) δ 162.6, 159.1, 150.7, 137.9, 137.2, 137.0, 130.7, 129.0, 128.7,
128.6, 128.4, 128.3, 128.1, 127.9, 113.7, 101.4, 89.2, 87.3, 83.6, 75.6,
73.7, 73.1, 70.0, 59.4, 55.3, 53.2, 43.5; HRMS (ESI) calcd for
C33H36N5O7 [M + H]+ 614.2615, found [M + H]+ 614.2609 (Δm
−0.0006, error −1.0 ppm).
1-[2′-O-Methyl-4′-C-trifluoroacetylaminomethyl-β-D-ribofur-
anosyl]-uracil (12). Compound 11 (3.5 g, 6.21 mmol) was dissolved
in EtOH (106 mL), and then 10% Pd/C (1.5 g) was added and heated
at 70 °C for 16 h under H2 atmosphere. The reaction mixture was
filtered through Celite pad and washed with MeOH (600 mL). The
MeOH layer was evaporated and purified by column chromatography
(5% MeOH in DCM) to give 12 as a white solid (1.65 g, 71%); Rf =
1-[2′-O-Methyl-4′-C-trifluoroacetylaminomethyl-β-D-ribofur-
anosyl]-3-(4-methoxybenzyl)-5,6-dihydrouracil (9). Nucleoside
8 (1.5 g, 2.4 mmol) was dissolved in dry MeOH (39 mL). To this
1
0.24, (5% MeOH in DCM); mp 124−126 °C; H NMR (300 MHz,
were added 20% Pd (OH) /C (2.19 g, 15.6 mmol) and ammonium
2
CD3OD) δ 8.04 (d, J = 8.1 Hz, 1H), 6.05 (d, J = 4.2 Hz, 1H), 5.7 (d, J
= 8.1 Hz, 1H), 4.51 (d, J = 5.7 Hz, 1H), 4.02 (dd, J = 5.7 Hz, 4.8 Hz,
1H), 3.67, 3.59 (ABq, J = 14.1 Hz, 2H), 3.65, 3.60 (ABq, J = 12.2 Hz,
2H), 3.51 (s, 3H); 13C NMR (75 MHz, CD3OD) δ 166.2, 159.5,
152.2, 142.8, 119.5, 115.7, 102.9, 89.2, 85.2, 71.2, 64.3, 59.3, 42.1; 19F
NMR (282 MHz, CD3OD) δ −76.1; HRMS (ESI) calcd for
C13H17F3N3O7 [M + H]+ 384.1019, found [M + H]+ 384.1028 (Δm
+0.0009, error +2.6 ppm).
formate (9.07 g, 144 mmol), and the reaction mixture was refluxed in
H2 atmosphere for 6 h. The reaction mixture was passed through a
Celite pad, washed with MeOH (500 mL), concentrated under
reduced pressure, and dried under high vacuum. The crude product
was dissolved in dry MeOH (100 mL), to this solution were added
CF3COOEt (0.94 mL, 7.92 mmol) and Et3N (1.83 mL, 13.2 mmol),
and the mixture was stirred at room temperature overnight. The
MeOH was evaporated under reduced pressure, and the residue was
purified by column chromatography (2% MeOH in DCM) to afford
sticky colorless compound 9 (680 mg, 71%); Rf = 0.51 (5% MeOH in
DCM); 1H NMR (400 MHz, CDCl3) δ 7.37−7.27 (m, 4 H), 6.82 (d, J
= 8.6 Hz, 2 H), 5.52 (d, J = 6.8 Hz, 1 H), 4.90, 4.85 (ABq, J = 14.0 Hz,
2 H), 4.40 (d, J = 5.6 Hz, 1 H), 4.18 (dd, J = 6.8 Hz, 5.8 Hz, 1 H), 3.78
(s, 3 H), 3.71− 3.46 (m, 6 H), 3.44 (s, 3H), 2.76−2.72 (m, 2 H); 13C
NMR (100 MHz, CDCl3) δ 168.9, 159.1, 158.5, 158.1, 153.9, 130.5,
129.6, 117.4, 114.6, 113.5, 90.3, 86.1, 80.1, 80.0, 71.0., 65.0, 58.9, 55.3,
43.6, 41.1, 38.8, 31.9. HRMS (ESI) calcd for C21H27N3O8F3 [M + H]+
506.1750, found [M + H]+ 506.1732 (Δm −0.0018, error −3.5 ppm).
1-[2′-O-Methyl-3′,5′-di-O-benzyl-4′-C-azidomethyl-β-D-ribo-
furanosyl]-uracil (10). Compound 8 (840 mg, 1.37 mmol) was
dissolved in a CH3CN/H2O mixture (17 mL, v/v 9:1), and ceric
ammonium nitrate (2.3 g, 5.5 mmol) was added. The reaction mixture
was stirred at room temperature for 55 h. Saturated aqueous NaHCO3
(100 mL) solution was added, and the mixture was stirred for an
additional 20 min, then filtered, and extracted with CH2Cl2 (3 × 125
mL). The extract was dried over Na2SO4 and evaporated under
reduced pressure. The residue was purified by column chromatog-
raphy (40% ethyl acetate in pet ether) to give pale yellow oily
1-[2′-O-Methyl-4′-C-trifluoroacetylaminomethyl-5′-O-(4,4′-
dimethoxytrityl)-β-D-ribofuranosyl]-uracil (13). Compound 12
(200 mg, 0.52 mmol) was co-evaporated with dry pyridine (2 × 10
mL) and redissolved in the same solvent (8 mL). DMT-Cl (352 mg,
1.04 mmol) was added and stirred at room temperature for 24 h. A
saturated solution of NaHCO3 (50 mL) was added and extracted with
DCM (3 × 80 mL). The organic layer was dried over Na2SO4, filtered,
and evaporated under reduced pressure. The residue was purified by
column chromatography (60% ethyl acetate in pet ether + 2% Et3N)
to give 13 as pale yellow solid (287 mg, 81%); Rf = 0.3, (60% ethyl
1
acetate in pet ether + 2% Et3N); mp 122−123 °C; H NMR (400
MHz, CDCl3) δ 7.61 (d, J = 8.1 Hz, 1H), 7.36− 7.23 (m, 10H), 7.09
(t, J = 5.8 Hz, 1H), 6.83 (d, J = 8.9 Hz, 4H), 6.04 (d, J = 4.2 Hz, 1H),
5.36 (d, J = 8.1 Hz, 1H), 4.52 (d, J = 6 Hz, 1H), 4.04 (dd, J = 5.7 Hz,
4.5 Hz, 1H), 3.79 (s, 6H), 3.64 (d, J = 6 Hz, 2H), 3.55 (s, 3H), 3.42,
3.32 (ABq, J = 10.7 Hz, 2 H); 13C NMR (100 MHz, CDCl3) δ 163.4,
159.0, 157.7, 150.4, 143.9, 140.3, 134.8, 134.6, 130.2, 128.3, 128.2,
127.5, 116, 113.5, 103.0, 87.9, 87.3, 83.8, 71.2, 65.6, 59.5, 55.2, 41.3;
19F NMR (282 MHz, CD3OD) δ −75.4 (s); HRMS (ESI) calcd for
C34H35F3N3O9 [M + H]+ 686.2325, found [M + H]+ 686.2354 (Δm
+0.0028, error +4.1 ppm).
1
compound 10 (517 mg, 76%); Rf = 0.26, (2% MeOH in DCM); H
NMR(300 MHz, CDCl3) δ 9.22 (s, 1H), 7.93 (d, J = 7.8 Hz, 1 H),
7.39−7.20 (m, 11 H), 6.03 (d, J = 1.5 Hz, 1 H), 5.14 (d, J = 8.1 Hz, 1
H), 4.72, 4.46 (AXq, J = 11.7 Hz, 2 H), 4.49, 4.42 (ABq, J = 11.1 Hz, 2
H), 4.27 (d, J = 5.4 Hz, 1 H), 3.95, 3.31 (AXq, J = 13.6 Hz, 2 H), 3.96,
3.55 (AXq, J = 10.5 Hz, 2 H), 3.77 (dd, J = 5.5, 1.5 Hz, 1 H), 3.56 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 163.6, 150.2, 140.4, 137.2,
137.1, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3, 128.1, 101.8, 88.7, 87.6,
83.7, 75.6, 73.9, 73.2, 70.1, 59.5, 53.3; HRMS (ESI) calcd for
C25H28N5O6 [M + H]+ 494.2040, found [M + H]+ 494.2060 (Δm
+0.0021, error +4.2 ppm).
1-[2′-O-Methyl-3′,5′-di-O-benzyl-4′-C-trifluoroacetylamino-
methyl-β-D-ribofuranosyl]-uracil (11). Nucleoside 10 (1.1 g, 2.23
mmol) was dissolved in THF (23 mL). To this were added water (0.2
mL) and PPh3 (1.17 g, 4.46 mmol). The reaction mixture was heated
at 45 °C for 4 h and then cooled to 0 °C. Then CF3COOEt (1.33 mL,
11.15 mmol) and Et3N (1.55 mL, 11.15 mmol) were added, and the
reaction mixture was stirred at room temperature for overnight. The
solvent was evaporated under reduced pressure and co-evaporated
with toluene (2 × 40 mL). The crude compound was purified by
column chromatography (40% ethyl acetate in pet ether) to afford
1-{2′-O-Methyl-3′-O-[2-cyanoethoxy(diisopropylamino)-
phosphino]-4′-C-trifluoroacetylaminomethyl-5′-O-(4,4′-dime-
thoxytrityl)-β-D-ribofuranosyl}-uracil (14). Compound 13 (150
mg, 0.22 mmol) was dissolved in DCM (3 mL). To this were added
DIPEA (0.3 mL, 1.74 mmol) and CEP-Cl (0.15 mL, 0.65 mmol), and
the mixture was stirred at room temperature for 75 min. Then MeOH
(0.5 mL) was added and stirred for 15 min. The reaction mixture was
diluted with DCM (100 mL), washed with NaHCO3 (3 × 50 mL),
dried over Na2SO4, and evaporated under reduced pressure. The crude
compound was purified by column chromatography (50−60% DCM
in pet ether) to give 14 as a white solid (130 mg, 70%); Rf = 0.7 (8%
MeOH in DCM + 2% Et3N); mp 80−81 °C; 31P NMR (162 MHz,
CDCl3) δ 151.40, 151.16; 19F NMR (282 MHz, CDCl3) δ −75.82,
−75.98; HRMS (ESI) calcd for C43H52N5O10F3P [M + H]+ 886.3404,
found [M + H]+ 886.3401 (Δm −0.0003, error −0.3 ppm).
1-[2′-O-Methyl-4′-C-trifluoroacetylaminomethyl-5′-O-(4,4′-
dimethoxytrityl)-β-D-ribofuranosyl]-cytosine (17). Nucleoside 13
(50 mg, 0.073 mmol) was dissolved in dry pyridine (2 mL). To this
was added Ac2O (0.03 mL, 0.36 mmol), and the mixture was stirred at
room temperature overnight. Then, NaHCO3 (30 mL) was added and
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extracted with EtOAc (3 × 60 mL). The EtOAc layer was dried over
Na2SO4, evaporated followed by co-evaporation with toluene (20 mL),
and dried under high vac to give crude compound 15. Compound 15
(50 mg, 0.073 mmol) was dissolved in DCM (1.5 mL) in an ice bath,
and then DMAP (1 mg, 0.008 mmol), Et3N (0.1 mL, 0.73 mmol) and
2,4,6-triisopropylbenzenesulphonylchloride (33 mg, 0.11 mmol) were
added. The reaction mixture was stirred at room temperature for 6 h.
Then the reaction mixture was diluted with DCM (70 mL), washed
with saturated NaHCO3 (3 × 60 mL), dried over Na2SO4, and
evaporated under reduced pressure. The crude compound 16 obtained
was dissolved in THF (1.6 mL), then aq NH3 (0.3 mL) was added at 0
°C, and the mixture was stirred at room temperature for overnight.
The reaction mixture was evaporated and purified by column
chromatography (5% MeOH in DCM + 2% Et3N) to give nucleoside
17 as pale yellow solid (31 mg, 63%, after 3 steps); Rf = 0.35 (4%
was 10 min, and for unmodified amidite (2′-O-TBMDS protected) it
was 6.5 min. 5-Ethylthio-1H-tetrazole (ETT) was used as coupling
agent. After the synthesis, the CPG beads were treated with the 400
μL of 10% diethyl amine in anhydrous acetonitrile to selectively
remove cyanoethyl groups. Then the beads were washed with
acetonitrile (2 × 400 μL). The supernatant was removed, and the
support was dried under desiccant mode in speed vac. The cleavage
from support and deprotection of base protecting groups were carried
out by treating the beads with 1 mL of 41% methylamine in water and
30% aq NH3 (1:1 v/v) for 18 min at 65 °C. Supernatant was collected,
and the support was washed with 1:1 mixture (300 μL) of EtOH and
water. The combined fractions were evaporated on a vacuum
concentrator. To remove 2′-O-TBDMS groups, oligonucleotides
were dissolved in 100 μL of dry DMSO, and 125 μL of Et3N·3HF
was added and heated at 65 °C for 2.5 h. The Et3N·3HF was quenched
with the addition of 400 μL of isopropyl trimethyl silyl ether, and RNA
was precipitated twice by adding 850 μL of cold diethyl ether. RNA
was recovered by centrifugation at 16,100 RCF for 10 min at 4 °C.
The RNA pellet was dried in the desiccant mode in a vacuum
concentrator for 20 min. The crude RNA was dissolved in water and
purified by 20% denaturing PAGE (7 M urea) at 30 W using 1× TBE
buffer (89 mM each Tris HCl and boric acid and 2 mM EDTA, pH
8.3). The gel was visualized, and bands were marked under UV lamp.
The desired gel band was excised and crushed, 15 mL of TEN (10 mM
Tris, 1 mM EDTA, 300 mM NaCl pH 8.0) was added, and the mixture
was shaken for 16 h at room temperature for the recovery. The RNA
samples were desalted using a Sep-Pak (C-18) column. After
evaporation, the RNA pellet was dissolved in water, and the
concentration was measured at 260 nm in a UV−vis spectropho-
tometer using appropriate molar extinction coefficients (ε).
Thermal Melting Experiments. Melting temperature analysis was
carried out by measuring change in absorbance at 260 nm with
increase in temperature using a UV spectrophotometer coupled with a
peltier controller. Duplexes were prepared using equimolar concen-
tration (1 μM) of complementary strands in a buffer solution
containing 100 mM NaCl, 10 mM Na2PO4 pH 7.4, 0.1 mM EDTA.
The solution containing passenger strand and guide strands was
heated at 95 °C for 2 min, then gradually cooled to room temperature,
and stored at 4 °C. Before the measurements, samples were
equilibrated at 25 °C for 10 min and then heated to 95 °C in an
increment of 0.5 °C/min. The change in absorbance against increase
in temperature was recorded, and Tm was calculated utilizing the first
order derivative method. All experiments were triplicated, and the Tm
values reported are average of 3 measurements.
Cell Culture and Gene Silencing Studies. HeLa cells were
grown in Eagle′s minimum essential media (MEM) supplemented with
10% heat inactivated fetal bovine serum (FBS) and the antibiotics
penicillin (100 units/mL) and streptomycin (100 units/mL) at 37 °C
in 5% CO2 environment. Twenty-four hours prior to transfection, cells
were plated in 24-well plates at a density of 3 × 10 5 cells/well. Before
transfection, the media was removed, and cells were washed with
phosphate buffered saline (PBS; 137 mM NaCl, 27 mM KCl, 10 mM
Na2HPO4, 2 mM KH2PO4, pH 7.4). The cells were transfected with
siRNA (33 nM, 10 pmol/well) in serum-free media. The cells were
cotransfected with psiCHECK2 plasmid (0.08 nM, 100 ng/well); 1.5
μL (1 μg/μL) of Lipofectamine-2000 was used for the transfection,
and the protocols detailed in the Lipofectamine-2000 manual were
followed. The cells were incubated for 5 h, and media was replaced
with fresh media containing 20% FBS without antibiotics. Then after
12 h, the media was changed with fresh media containing 20% FBS
with antibiotics. After 48 h post transfection, media was removed, and
the cells were washed with PBS and then lysed using passive lysis
buffer. The cell lysate was used for dual luciferase assay system and
gene expression measured using luminometer. The ratio between
relative light units of Renilla and Firefly luciferase was determined for
each well and normalized using the value obtained from positive
control experiments (no siRNA). The luminescence values reported
are an average of four independent experiments. Statistical analyses
between individual groups were performed by a Student t test; a P
value of <0.05 was considered statistically significant.
1
MeOH in DCM + 2% Et3N); mp 124−126 °C; H NMR(400 MHz,
CD3OD) δ 7.97 (d, J = 7.5 Hz, 1H), 7.41 (m, 2 H), 7.35−7.24 (m,
7H), 6.86 (d, J = 8.5 Hz, 4H), 6.04 (d, J = 2.3 Hz, 1H), 5.43 (d, J = 7.4
Hz, 1H), 4.71 (d, J = 6 Hz, 1H), 3.88 (dd, J = 6 Hz, 2.5 Hz, 1H), 3.77
(s, 6H), 3.69, 3.62 (ABq, J = 14.0 Hz, 2 H), 3.57 (s, 3H), 3.49−3.44
(m, 2H); 13C NMR (100 MHz, CD3OD) δ 167.7, 160.4, 160.3, 157.9,
145.9, 143.0, 136.7, 136.4, 131.6, 131.5, 129.5, 129.0, 128.2, 116.9,
114.3, 114.3, 96.1, 90.7, 88.7, 88.5, 86.0, 71.4, 65.0, 59.7, 55.8, 53.8,
42.4; 19F NMR (282 MHz, CD3OD) δ −77.0; HRMS (ESI) calcd for
C34H36N4O8F3 [M + H]+ 685.2485, found [M + H]+ 685.2485, (Δm
0.0, error 0.0 ppm).
1-[2′-O-Methyl-4′-C-trifluoroacetylaminomethyl-5′-O-(4,4′-
dimethoxytrityl)-β-D-ribofuranosyl]-N4-(isobutyryl)-cytosine
(18). Nucleoside 17 (70 mg, 0.10 mmol) was dissolved in dry pyridine
(2 mL). Then trimethylsilyl chloride (0.06 mL, 0.51 mmol) was added
dropwise at 0 °C and stirred at room temperature for 45 min.
Isobutyryl chloride (0.02 mL, 0.20 mmol) was added at 0 °C and
stirred for 4 h. After completion of reaction, dry MeOH (0.5 mL) was
added and stirred for 20 min. Saturated NaHCO3 solution (50 mL)
was added and extracted with DCM (3 × 60 mL). The organic layer
was dried over Na2SO4 and evaporated under reduced pressure. The
crude compound was purified by column chromatography (2% MeOH
in DCM + 2% Et3N) to give 18 as white solid (50 mg, 67%); Rf = 0.44
(3−4% MeOH in DCM + 2% Et3N); mp 164−166 °C; 1H NMR (400
MHz, CDCl3) δ 8.66 (bs, 1H), 8.06 (d, J = 7.5 Hz, 1H), 7.38−7.23
(m, 9H), 7.10 (d, J = 7.5 Hz, 1H), 6.85−6.81 (m, 4H), 6.08 (d, J = 2.2
Hz, 1H), 4.50 (d, J = 6.1 Hz, 1H), 3.99−3.93 (m, 1H), 3.80 (s, 6H),
3.78−3.66 (m, 2H), 3.64 (s, 3H), 3.44, 3.38 (ABq, J = 10.7 Hz, 2H),
2.60 (sep, J = 6.8 Hz, 1H), 1.20 (d, J = 6.8 Hz, 3H); 1.19 (d, J = 6.8
Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 177.1, 162.8,158.9, 157.7,
157.5, 155.2, 144.7, 143.8, 135.2, 134.8, 130.3, 130.1, 128.3, 128.2,
127.5, 116, 113.6, 97.0, 89.9, 87.7, 87.7, 84.6, 70.8, 65.2, 59.8, 55.4,
41.2, 36.7, 19.2, 19.1; 19F NMR (376 MHz, CDCl3) δ −75.82; HRMS
(ESI) calcd for C38H42N4O9F3 [M + H]+ 755.2904, found [M + H]+
755.2905 (Δm +0.0001, error +0.1 ppm).
1-{2′-O-Methyl-3′-O-[2-cyanoethoxy(diisopropylamino)-
phosphino]-4′-C-trifluoroacetylaminomethyl-5′-O-(4,4′-dime-
thoxytrityl-β-D-ribofuranosyl}-N4-(isobutyryl)-cytosine (19).
The compound 18 (50 mg, 0.06 mmol) was dissolved in DCM (1.2
mL). Then DIPEA (0.09 mL, 0.52 mmol) and CEP-Cl (0.04 mL, 0.2
mmol) were added and stirred at room temperature for 40 min. After
completion of reaction, dry MeOH (0.05 mL) was added and stirred
for 15 min. The reaction mixture was diluted with DCM (100 mL),
washed with saturated NaHCO3 (3 × 50 mL), dried over Na2SO4, and
evaporated under reduced pressure. The crude compound was purified
by column chromatography (60% DCM in pet ether) to give 19 as
white solid (35 mg, 51%); Rf = 0.88 (4% MeOH in DCM + 2% Et3N);
mp 94−95 °C; 31P NMR (162 MHz, CDCl3) δ 151.18, 151.10; 19F
NMR (282 MHz, CDCl3) δ −76.25, −76.31; HRMS (ESI) calcd for
+
C47H59N6O10F3P [M + H]+ 955.3982, found [M + H] 955.4011
(Δm +0.0028, error +3.0 ppm).
RNA Synthesis and Purification. The unmodified and modified
siRNA sequences were synthesized on 0.2 μmol scale on an automated
DNA/RNA synthesizer using standard CPG solid support. The
coupling time used for modified uridine and cytidine phosphoramidite
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5′-End Radiolabeling of Oligonucleotides. Twenty-five
picomoles of RNA was mixed with PNK enzyme (5 units) and γ-32P
ATP (10 μCi) in PNK buffer (50 mM Tris-HCl, 10 mM MgCl2, 5
mM DTT, 0.1 mM each spermidine and EDTA, pH 7.6) with a final
volume of 10 μL. The reaction mixture was incubated at 37 °C for 1 h.
The enzyme was deactivated by the addition of 10 μL of stop solution
(80% formamide, 50 mM EDTA, 0.025% of each bromophenol blue
and xylene cyanol). The labeled oligonucleotides were purified by 20%
denaturing PAGE (7 M urea), extracted with TEN, and precipitated
using ethanol.
the backbone (C3′, O3′, P, O1P, O2P, C5′ and O5′) of each
nucleotide.
The percentage of occurrence of C3′-endo sugar conformation
during MD simulation at each nucleotide in both strands of all of the
duplexes were calculated using the X3DNA program. The amplitude of
the pseudo rotational angle (tm), which represents the four dihedral
angles with their distribution, was determined for each nucleotide in all
duplexes using the X3DNA program. Water density or hydration
numbers, which represent the average number of water molecules
present in a distance of less than 3.0 Å from solute atoms over 20 ns,
were computed for major groove, minor groove and backbone atoms
separately using Ptraj module in AMBER 10. The helicoidal
deformation parameters (buckle, open angle, propeller, stagger,
shear, stretch)87 and base pair step parameters (shift, slide, roll, rise,
tilt, twist)88 were calculated for each dinucleotide steps of all of the
duplexes by the X3DNA program. The distribution of χ angle at the
modified nucleotide level was calculated using the Ptraj module and fit
to normalize Gaussian. The percentages of occupancies of two
Watson−Crick hydrogen bonds between A-U base pair at the modified
position were also determined over 20 ns simulation using Ptraj
module.
The stacking and hydrogen bond interaction energies at the
modified regions were computed at the MP2(full)/6-311G* level
using the Gaussian03 program.53 The interaction energy was
categorized into hydrogen bond energy (ΔEHB), interstrand stacking
energy (ΔEinter), and intrastrand stacking energy (ΔEintra). Stacking
energies were calculated solely based upon the electrostatic and van
der Waals interaction, which were calculated using Molecular
Mechanics with AMBER FF0389 and Lennard−Jones (6−12)
potential. Basis set superposition error (BSSE) for hydrogen bonds
energy and stacking interactions were corrected using the counterpoise
method.90 All of the structures were visualized, and the figures were
rendered using PyMOL v0.99 (http://www.pymol.org).
Serum Stability Assay. 32P-Labeled guide strands (0.15 μM) were
mixed with excess cold complementary unmodified passanger strands
(1 μM) in PBS. The solutions were heated at 95 °C for 2 min and then
gradually cooled to room temperature. The annealed siRNA duplexes
were incubated at 25 °C in 10% human serum (from male AB clotted
whole blood) in PBS with a final volume of 10 μL. The aliquots of 1
μL were withdrawn after 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 22 h,
and 44 h, quenched with 4 μL of stop solution (80% formamide, 50
mM EDTA, 0.025% of each bromophenol blue and xylene cyanol),
and stored at −20 °C. The samples were analyzed on 20% denaturing
PAGE (7 M urea). Gels were visualized by using a phosphorimager.
Molecular Modeling Studies. The starting structures of three
isosequential siRNA duplexes (3R-2R, 3R-7R, and 6R-7R) with
modifications at different positions were modeled using the NUCGEN
module implemented in AMBER 10.54 The force field parameters for
the modified nucleotides were obtained using the protocol developed
by Aduri and co-workers,81 and RESP charges were derived using
Antechamber RESP fitting procedure developed by Kollman and co-
workers.82 Unmodified uridine nucleotide at respective positions were
replaced by 4′-C-aminomethyl-2′-O-methyl modified uridine using the
calculated parameters, and charges by XLeap module in AMBER 10.
Each duplex was immersed in an octahedron-shaped box containing
∼4500 TIP3P water molecules extended up to 10 Å away from any of
the solute atom. Forty-one sodium ions were randomly added to
neutralize the charge of the phosphate backbone. All of the simulations
reported here were performed using the SANDER module of AMBER
10. After solvation, the system was then subjected to an equilibration
protocol involving 2500 steps of steepest descent minimization of
solvent, and 2500 steps of conjugate gradient, holding the solute/RNA
duplex to their initial position by means of positional restraints.
Further minimization was carried for the whole system by 5000 steps
of conjugate gradient method without any positional restraints,
allowing the system to attain relaxed geometry. Then the solvent
was heated from 0 to 300 K, with 10 kcal/(mol Å2) constraints for
RNA duplex in a period of 100 ps keeping a constant volume. Next the
equilibration was continued to 200 ps at a constant pressure (1 atm)
and temperature (300 10 K). Constant temperature was maintained
using a Langvein algorithm. After equilibration, systems were subjected
to 20 ns of MD simulation saving trajectory coordinates for each
picosecond. All covalent bonds involving hydrogen atoms were
constrained using SHAKE.83 Long range interactions were treated
using the particle mesh Ewald method,84 and the cut-off distance was
set to10 Å for Lennard−Jones potential.
ASSOCIATED CONTENT
* Supporting Information
■
S
Thermal melting plots for modified siRNAs, additional data
from molecular modeling studies, ESI-MS data for oligonucleo-
tides, and copies of NMR spectra of all the new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: pradeep@chem.iitb.ac.in.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are thankful to Computer Centre, IIT Bombay and BRAF-
CDAC, Pune for providing computing facility and SAIF-IIT
Bombay for NMR spectra. We are also thankful to V.
Dhamodharan for critically reading the manuscript; Prof.
David A. Case for waiving of the licensing fee for AMBER
10; Akshay Tanna for the assistance with the RNA synthesis;
Prof. K.P. Kaliappan for providing access to the melting point
apparatus; and Dr. Prasenjit Mitra, ILS-Hyderabad for the help
with the cell culture experiments. This work is financially
supported by grants from Department of Biotechnology
(DBT)-Government of India (RNAi-Technologies Platform,
BT/PR10693/AGR/36/586/2008); Council of Scientific and
Industrial Research-Government of India (CSIR, 01-2233/08/
EMR-II); Department of Science and Technology (DST)-
Government of India (FAST track scheme, SR/FT/LS-133/
2008); and IRCC-IIT Bombay. P.I.P. is a recipient of Max-
Planck India Fellowship (MPG-DST scheme). K.R.G. thanks
Trajectory analysis was done using the Ptraj module in AMBER 10,
X3DNA program,85 in-house programs and examined visually using
VMD 1.4.6.86 The rotational and translational motions were removed
from trajectories by superimposing the siRNA duplex to their starting
structure. The root-mean-square deviation (rmsd) values were
calculated over 20 ns MD simulation for all heavy atoms in each
duplex with respect to their corresponding reference structure. The
rmsd values were also calculated for the regions containing modified
nucleotides flanked by one base pair on either side along with their
complementary residues. The interstrand phosphate distance was
calculated as the distance between the phosphorus atom of a
nucleotide in one strand and phosphorus atom of a nucleotide in
the partner strand, and the intrastrand phosphate distance as the
distance between phosphorus atoms in two successive nucleotides
within a strand. The root-mean-square fluctuation (rmsf) values were
calculated around average structures for the heavy atoms composing
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University Grants Commission (UGC) for a fellowship. G.N.N.
thanks CSIR and German Academic Exchange Program
(DAAD) for fellowships.
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