Linear relationship between deformability and thermal stability of 2'-O-modified RNA hetero duplexes

Article abstract of DOI:10.1021/jp909851j

We describe the relationship between the experimentally determined melting temperatures of 2'-O-modifiedRNA/RNA duplexes and their deformability estimated from molecular dynamics simulations. To clarify this relationship, we synthesized several fully modi

Full text of DOI:10.1021/jp909851j

J. Phys. Chem. B 2010, 114, 2517–2524
2517
Linear Relationship between Deformability and Thermal Stability of 2′-O-Modified RNA
Hetero Duplexes
Yoshiaki Masaki, Ryuta Miyasaka, Akihiro Ohkubo, Kohji Seio, and Mitsuo Sekine*
Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Japan
ReceiVed: October 14, 2009; ReVised Manuscript ReceiVed: NoVember 19, 2009
We describe the relationship between the experimentally determined melting temperatures of 2′-O-modified-
RNA/RNA duplexes and their deformability estimated from molecular dynamics simulations. To clarify this
relationship, we synthesized several fully modified oligoribonucleotides such as 2′-O-cyanoethyl RNAs and
2′-O-methoxyethyl RNAs and compared the actual melting temperatures of the duplexes with their calculated
deformabilities. An increase of the melting temperatures by 2′-O-modifications was found to correlate strongly
with an increase of the helical elastic constants in U14/A14, (CU)7/(AG)7, and (GACU)3/(AGUC)3 sequences.
Linear regression analyses could be used to estimate the melting temperature with an accuracy of (2.0 °C in
our model case. Although the strong correlation was observed in the same base sequence, the linear regression
functions were different from each base sequence. Our results indicated the possibility of predicting the thermal
stability of 2′-O-modified duplexes at the computer-aided molecular design stage.
Introduction
Chemical modification of natural RNAs has been of great
importance in improving their original physicochemical and
biochemical properties. Since RNAs are rapidly degraded by
cellular nucleases, a large number of 2′-O-modified RNAs have
been developed and applied to the gene regulation in antisense,
antigene, and RNA interference (RNAi) strategies.1-5 We are
interested in 2′-O-alkyl-type modifications because they can
enhance both hybridization affinity and the nuclease resistances.
In contrast, it is reported that other modifications such as 2′-F
modification increased the hybridization affinity but not enhance
the nuclease resistance in comparison with unmodified DNA.6
As one such 2′-O-modified RNA, we reported 2′-O-cyanoet-
hylated RNA that showed enhanced hybridization affinity and
nuclease resistance compared with 2′-O-methyl RNA.7,8
Currently, there is no method of predicting the hybridization
properties of 2′-O-modified-RNAs, and they can be determined
only experimentally, using actually synthesized 2′-O-modified-
RNA samples. However, comparative studies among various
2′-O-modified RNAs are, in general, laborious and time-
consuming tasks because it is sometimes difficult to synthesize
certain 2′-O-modified nucleosides. Thus, a facile computational
method for predicting thermal stability is eagerly anticipated
for the efficient design of new 2′-O-modified-RNAs.
The effect of 2′-O-modification on the stability of RNA
duplexes has been studied especially for the 2′-O-methyl
group.9-20 The stabilizing effect by the 2′-O-methyl group was
commonly explained by the rigidity of the sugar puckering.9-11
The rigid sugar puckering suppresses the entropy loss to form
a duplex from the single strand.21 1H NMR studies showed that
the 2′-O-methylation of dinucleoside monophosphate derivatives
increased the population of the C3′-endo form except for
adenosine derivatives.10 Circular dichroism (CD) studies also
showed that the 2′-O-methylation increased the intensity of CD
spectra in the dinucleoside monophosphates except for the
adenine derivatives.15,16
Figure 1. Pictorial definitions of parameters that relate sequential base-
pair steps. The parameter values were calculated using the X3DNA
software package.40
However, sugar puckering is not the ideal indicator. Although
2′-O-cyanoethylation apparently increased the Tm values of
modified RNA duplexes more than 2′-O-methylation as reported
by us, little difference was found in the population of the C3′-
endo form between the 2′-O-cyanoethyl and 2′-O-methyl
1
nucleosides by our H NMR analysis.7
Recently, in addition to these conformational effects, the
hydration shell has also been considered to play an important
role in the 2′-O-methyl RNA/2′-O-methyl RNA homo duplex
stability.17,18
The concept of deformability was widely used to evaluate
the thermodynamic properties of nucleic acids, such as nucleo-
some folding, elasticity of rRNA, and RNaseH susceptibility.22-30
De Santis and co-workers reported that the relative rigidities of
several kinds of base pair steps in DNA and normalized melting
temperatures are strongly correlated.28-30 However, no research
has estimated the effect of 2′-O- modification of RNAs by using
the deformability.
In this paper, we show the relationship between the melting
temperatures (Tm) of duplexes (U14/A14, (CU)7/(AG)7, and
(GACU)3/(AGUC)3) and their deformabilities (sugar puckering,
twist, roll, tilt, rise, shift, and slide) that are inferred from
molecular dynamic simulations (Figure 1). The chemical
structures of 2′-O-modified nucleosides used in this study are
* Corresponding author. Tel.: +81-45-924-5706. Fax: +81-45-924-5772.
E-mail address: msekine@bio.titech.ac.jp.
10.1021/jp909851j  2010 American Chemical Society
Published on Web 01/28/2010

2518 J. Phys. Chem. B, Vol. 114, No. 7, 2010
Masaki et al.
the highest Tm of 43 °C. Interestingly, the effect of 2′-O-
modification of the adenosine residues was clearly different from
that of uridine residues. The Tm values of UOH14/AOH14, UOH
/
14
AOMe14, and UOH14/AOCE14 were 24, 24, and 27 °C, respectively.
In this case, the 2′-O-methylation had only negligible effects
on the Tm value, and the CE modification also gave a small Tm
increase of 3 °C.
Sugar Puckering. The rigidity of sugar puckering has
frequently been used to explain the effect of 2′-O-modification.9-11
We tried to clarify the relationship between the rigidity of sugar
puckering and the thermal stability of duplexes. The histograms
of pseudorotation angles of the modified or unmodified uridine
residues in UOH14/AOH14, UOMe14/AOH14, UOMOE14/AOH14, and
Figure 2. Chemical structures of 2′-O-modified nucleotides.
UOCE14/AOH duplexes and the modified and unmodified ad-
14
enosine residues in UOH14/AOH14, UOH14/AOMe14, and UOH14/AOCE
14
duplexes are shown in Figure 3.
shown in Figure 2. We demonstrated that the deformability of
the rise parameter correlates well with the melting temperature.
This result suggested that the rigidity of the rise parameter, rather
than the frequently discussed sugar puckering, could be a good
indicator of the thermal stability changed by 2′-O-modification.
Our results also showed a stronger correlation between the total
stiffness indexes and the experimental Tm values. This means a
new possibility of predicting the thermal stability of 2′-O-
modified RNA/RNA duplexes at the molecular design stage.
As shown in Figure 3a, the pseudorotation angles of the 2′-
O-modified uridines, such as UOMe, UOMOE, and UOCE, are
consistently distributed more sharply than the unmodified uridine
residues. However, there was a negligible difference among the
2′-O-modified uridines. This tendency was also seen for the
adenosine residues, as shown in Figure 3b. The sharpness of
the distribution indicates the rigidity of the sugar puckering.
These results suggested that the 2′-O-modification rigidified the
sugar puckering markedly.
The rigidity was quantitatively represented by calculating the
elastic constants of sugar puckering. The calculated elastic
constants of UOH, UOMe, UOCE, and UOMOE were 3.5, 8.5, 8.6,
and 8.5 cal ·mol-1 ·deg-2, respectively (Table 1). Larger elastic
constant values indicate more rigid structures. Thus, the rigidities
of sugar puckering were increased by the 2′-O-methylation,
methoxyethylation, and cyanoethylation. As mentioned above,
there was only a negligible difference among the 2′-O-modified
uridines. This tendency was also observed for the elastic
constants of the adenosine residues. The elastic constants of
the pseudorotation angle of AOH, AOMe, and AOCE were 4.4, 7.7,
and 7.6 cal ·mol-1 ·deg-2, respectively.
Results
Synthesis of 2′-O-Modified Oligoribonucleotides. First, we
synthesized oligoribonucleotides having 2′-O-modified nucleo-
side residues such as 2′-O-cyanoethyluridine (UOCE), 2′-O-
methoxyethyluridine (UOMOE), and 2′-O-cyanoethyladenine
(AOCE) to compare their thermal stabilities. The phosphoramidite
of UOCE was synthesized by the ring-opening reaction of 2,2′-
anhydrouridine under BF3 ·OEt2, followed by tritylation and
phosphorylation.8 The phosphoramidite building block of UOMOE
was synthesized by Reese’s method.42 The phosphoramidite
building block of AOCE was prepared by the previously reported
procedure.7
Next, we compared these elastic constants and the experi-
mentally determined Tm values. A comparison of the order of
the elastic constants and the Tm values suggested that these two
parameters did not correlate. For example, the elastic constant
of AOMe (7.7 cal·mol-1 ·deg-2) was slightly larger than that of
AOCE (7.6 cal·mol-1 ·deg-2). However, the Tm value of AOMe
The oligoribonucleotide synthesis of UOMOE and UOCE was
carried out with universal support III (from Glen Research
Corp.) and a succinyl linker, respectively. The synthesis of 2′-
O-cyanoethyluridine-loaded CPG resins was reported previ-
ously.7 To avoid the elimination of the 2′-O-cyanoethyl group,
the synthesis of an AOCE oligomer was carried out using a silyl
linker,43 which can be cleaved under mild conditions, such as
0.2 M Et3N-3HF, as shown in Scheme 1. The 2′-O-cyanoet-
hyladenosine loaded CPG resin 4 was synthesized by the
silylation of compound 1 followed by the deprotection of the
fluorenylmethyl group and condensation with amino-loaded
CPG by treatment with DCC. Each chain elongation was
performed according to the standard procedure of RNA syn-
thesis. Purification of the crude products by anion exchange
(UOH14/AOMe14, 24 °C) was lower than that of AOCE (UOH
/
14
AOCE14, 27 °C).
Our results indicated that the rigidity of the sugar puckering
in the duplex inferred from molecular dynamics did not correlate
well with Tm values, at least in the duplexes having 2′-O-
modification.
Relationship between Tm Values and Elastic Constants of
Each Helical Parameter. Then, we analyzed the deformability
of the duplexes at the base pair step level. To quantify the
deformabilities, we carried out the harmonic stiffness analysis
introduced by Lankas et al. and Olson et al.22-24 The detailed
data are shown in Table S1 (Supporting Information).
HPLC gave the homo-oligoribonucleotides (14mer) UOCE
,
14
UOMOE14, and AOCE14. The 2′-O-unmodified oligoribonucleotides,
UOH14 and AOH14, and the 2′-O-methylated oligoribonucleotides,
UOMe14, AOMe14, (CU)OMe7, and (AG)OMe were purchased as
7
materials purified by HPLC (from Fasmac Co. Ltd.).
UV Melting Temperature Analysis. Next we measured the
melting temperature (Tm) of the duplexes. The duplexes of
The plots of the Tm values versus the elastic constants of the
six base step parameters showed linear correlations with positive
slopes in all cases (Figure 4). These results indicate that more
rigid duplexes show higher thermodynamic stabilities. The
correlation coefficients for the three translational parameters
(rise, shift, and slide) were 0.97, 0.94, and 0.74, respectively,
and the other three rotational parameters (twist, roll, and tilt)
were 0.89, 0.89, and 0.86, respectively. Among these, the
deformability of the rise parameter strongly correlated (R2 )
UOH14/AOH14, UOMe14/AOH14, UOMOE14/AOH14, and UOCE14/AOH
14
gave Tm values of 24, 36, 40, and 43 °C, respectively (Table
1). The methylation of the 2′-OH group of uridine increased
the Tm from 24 to 36 °C (by 12 °C). The introduction of the
methoxyethyl (MOE) group gave a much higher Tm of 40 °C,
and the cyanoethyl (CE) modified duplex UOCE14/AOH gave
14

Deformability of 2′-O-Modified RNA Hetero Duplexes
J. Phys. Chem. B, Vol. 114, No. 7, 2010 2519
SCHEME 1: Preparation of 2′-O-Cyanoethyladenosine-Loaded CPG Resin 4a
a Reagents and conditions: (a) 2, imidazole, CH2Cl2, rt, 1.5 h; (b) DBU, MeCN, rt, 15 min; (c) amino-loaded CPG, DCC, DMAP, CH2Cl2, rt,
24 h.
TABLE 1: Elastic Constants of Pseudorotation Angles and Tm Values
fsugar (cal·mol-1 ·deg-2
)
Tm (°C)
fsugar (cal·mol-1 ·deg-2
)
Tm (°C)
UOH
3.5a
8.5b
8.6c
8.5d
24
36
43
40
AOH
AOMe
AOCE
4.4a
7.7e
7.6f
24
24
27
UOMe
UOCE
UOMOE
a Data were extracted from the UOH14/AOH duplexes. b Data were extracted from the UOMe14/AOH duplexes. c Data were extracted from the
14
14
UOCE14/AOH duplexes. d Data were extracted from the UOMOE14/AOH duplexes. e Data were extracted from the UOH14/AOMe duplexes. f Data
14
14
14
were extracted from the UOH14/AOCE duplexes.
14
Figure 3. Histograms of pseudorotation angles. (a) Modified or unmodified uridine residues in UOH14/AOH14, UOMe14/AOH14, UOMOE14/AOH14, and
UOCE14/AOH duplexes. (b) Modified or unmodified adenosine residues in UOH14/AOH14, UOH14/AOMe14, and UOH14/AOCE duplexes.
14
14
0.97) with the Tm values. This observation suggests that the rise
parameter is the most useful indicator for predicting the Tm value
as long as each helical parameter is considered separately.
Relationship between Tm Values and Global Elastic
Constants of Helical Parameters. Next, we calculated the
elastic constants of translation (Ftrans), rotation (Frot), and their
product deformability (Fprod ) Ftrans × Frot) and compared them
with the Tm values. Detailed data are shown in Table S2
(Supporting Information). The plot of Tm values showed strong
correlation with Ftrans, Frot, and Fprod constants (Figure 5). The
correlation coefficients of Ftrans, Frot, and Fprod are 0.96, 0.92,
and 0.98, respectively. Among these three coefficients, Fprod had
the largest correlation efficient.
The strong correlation of Fprod with Tm values indicates the
importance of considering the product deformability for estima-
tion of the thermal stability. To predict the thermal stability
using this correlation, we calculated the linear approximation
equation by the least-squares method. The linear approximation
equation of Fprod and Tm values is given by
This equation can be used only for the A14/U14-type duplex
discussed in this paper. The data of the experimental and
predicted Tm values are shown in Table 2. The calculated Tm
values (and experimental Tm in parentheses) were 22.0 °C
(24 °C) for UOH14/AOH14, 35.0 °C (36 °C) for UOMe14/AOH
40.1 °C (40 °C) for UOMOE14/AOH14, 43.2 °C (43 °C) for UOCE
,
14
/
14
AOH14, 24.7 °C (24 °C) for UOH14/AOMe14, and 28.7 °C (27 °C)
for UOH14/AOCE14. The discrepancies between experimental and
the calculated data were -2.0 to +1.7 °C for the A14/U14-type
duplexes.
Deformability Analyses for Duplexes Having Mixed Se-
quences. Next, we studied the relationship that more rigid
structures showed higher Tm values by using 2′-O-modified
hetero duplexes having mixed sequences. For the analyses of
mixed sequences we tried to parametrize the deformability (Fprod
)
of each base-pair steps, 5′-N1N2-3′/3′-N3N4-5′, where N and N
represented either 2′-O-modified or unmodified nucleotide
residues. For example, for the parametrization of the 2′-O-
methyl-RNA/RNA duplex we have to obtain sixteen parameters
of the 5′-N1N2-3′/3′-N3N4-5′ base steps, where N1 and N2
Tm ) 3957 × Fprod + 17.4
(1)

2520 J. Phys. Chem. B, Vol. 114, No. 7, 2010
Masaki et al.
Figure 4. Correlations between the Tm values versus elastic constant of each helical parameter. The circles refer to UOH14/AOH (white), UOMe
/
14
14
AOH (red), UOMOE14/AOH (orange), UOCE14/AOH (purple), UOH14/AOMe (blue), and UOH14/AOCE14( green).
14
14
14
14
Figure 5. Correlations between Tm values versus Ftrans, Frot, or Fprod values. The circles refer to UOH14/AOH14 (white), UOMe14/AOH14 (red), UOMOE
/
14
AOH (orange), UOCE14/AOH (purple), UOH14/AOMe (blue), and UOH14/AOCE (green).
14
14
14
14
TABLE 2: Comparison of Experimental and Calculated Tm Values
Fprod [(kcal·mol-1 ·deg-1 ·Å-1)6]
Tm(exp) (°C)
Tm(calc) (°C)
∆Tm (°C)
a
duplex
UOH14/AOH
0.00116 ( 0.00024
0.00445 ( 0.00026
0.00573 ( 0.00026
0.00651 ( 0.00034
0.00185 (0.00012
0.00286 ( 0.00009
24
36
40
43
24
27
22.0
35.0
40.1
43.2
24.7
28.7
-2.0
-1.0
0.1
0.2
0.7
14
UOMe14/AOH
14
UOMOE14/AOH
UOCE14/AOH
14
14
UOH14/AOMe
14
14
UOH14/AOCE
1.7
a ∆Tm ) Tm(calc) - Tm(exp).
represent A, U, G, or C and N3 and N4 represented 2′-O-methyl-
A, -U, -G, or -C.
U)OMe3/(AGUC)OH3, (GACU)OCE3/(AGUC)OH3) were plotted
against the experimentally determined Tm values (Figure 6). The
detailed Ftotal, calculated and experimental Tm values are shown
in the Supporting Information (Table S5).
Surprisingly, even in the mixed sequences, the Tm values were
found to correlate well with the Ftotal values, which were only
the sums of the Fprod value of each base-pair step. The correlation
coefficient of a GACU sequence was 0.97 and that of a CU
sequence was 0.99. Thus, the deformability analyses could be
expanded to the duplexes with mixed sequences by introducing
To obtain the parameters, we carried out the MD simulations
and the deformability analyses by using Seq1: (UUAA)5UUA/
(UAAU)5UAA, Seq2: (CCGG)5CCG/(CGGC)5CGG, and Seq3:
(ACUG)5ACU/(AGUC)5AGU base sequences. By using the data
for Seq1, we can obtain the Fprod of 5′-AA-3′/3′-UU-5′, 5′-AU-
3′/3′-UA-5′, 5′-UA-3′/3′-AU-5′, and 5′-UU-3′/3′-AA-5′. In
addition, by using Seq2 and Seq3, we can obtain the Fprod for
all base pair steps. We defined a new parameter “total stiffness
index (Ftotal)” as the sum of Fprod. The calculated Fprod value of
each base pair step was shown in Table S4 (Supporting
Information).
new parameter Ftotal
.
Discussion
To verify the linear assumption between the melting tem-
perature and the total stiffness index (Ftotal), the Ftotal values of
In this paper we presented the deformability analysis for U14/
A14 and mixed sequences. These results indicated that deform-
ability is a good indicator for the estimation of the thermal
14mer duplexes ((CU)OH7/(AG)OH7, (CU)OMe7/(AG)OH7, (CU)OH
/
7
(AG)OMe7) and 12mer duplexes ((GACU)OH3/(AGUC)OH3, (GAC-

Deformability of 2′-O-Modified RNA Hetero Duplexes
J. Phys. Chem. B, Vol. 114, No. 7, 2010 2521
Despite the lack of rationale for the correlation, it is clear that
the deformability analysis is able to estimate the relative stability
of RNA duplexes incorporating 2′-OH, OMe, OCE, and OMOE
derivatives of ribonucleosides. The deformability analysis was
still not a perfect indicator of the predicting the thermal stability
of 2′-O-modified duplexes, but it showed possibility of predict-
ing the thermal stability at the computer-aided molecular design
stage.
Conclusion
We introduced two parameters (Fprod and Ftotal) and found
strong correlations with the melting temperatures. The correla-
tion would be used for the linear prediction of the Tm values
within (2.0 °C. We showed here the possibility of predicting
a change in thermal stability by 2′-O-modification. In addition,
this deformability analysis can also be used for biologically
useful 2′-O-modified designs having an appropriate thermal
stability.
Figure 6. Correlations between Tm values versus Ftotal values in
(GACU)3/(AGUC)3 and (CU)7/(AG)7 sequence. The data of CU
sequence are (CU)OH7/(AG)OH7, (CU)OMe7/(AG)OH7, and (CU)OH
/
,
7
(AG)OMe7. The data of GACU sequence are (GACU)OH3/(AGUC)OH
3
(GACU)OMe3/(AGUC)OH3, and (GACU)OCE3/(AGUC)OH (Table S5,
3
Supporting Information).
Experimental Section
1
stability of 2′-O-modified RNAs. Although the deformability
predicted the Tm values well, the contributions of the physico-
chemical factors such as hydrogen bonds, stacking interactions,
conformational rigidity, and hydration effects to the deform-
abilities have not been well understood. However, some
plausible relationships between the physicochemical factors and
deformabilities could be hypothesized. As previously reported,
the conformational effect of sugar puckering on uridine nucle-
otide by 2′-O-methylation is commonly explained by the steric
repulsion between the 2-carbonyl group and the 2′-O-methyl
group.9 The weak effect of 2′-O-methylation on the fluctuation
of the adenine base could be explained by the lack of this type
of steric repulsion. In the case of the sequences incorporating
2′-O-methyl-pyrimidine nucleosides, the rigidity of the sugar
conformation might reflect the large Ftotal value.
General Remarks. H spectra were recorded at 500 MHz
NMR. The chemical shifts were measured from CDCl3 (7.26
ppm). UV spectra were recorded with a U-2000 spectrometer.
Column chromatography was performed with silica gel C-200
(purchased from Wako Co. Ltd.). High performance liquid
chromatography (HPLC) was performed using the following
systems: Reversed exchange HPLC was done on a Waters
Alliance system with a Waters 3D UV detector and a Waters
XTerra MS C18 column (4.6 × 150 mm); a linear gradient
(0-30%) of solvent I [0.1 M ammonium acetate buffer (pH
7.0)] in solvent II (CH3CN) was used at 50 °C at a flow rate of
1.0 mL/min for 30 min; anion-exchange HPLC was done on a
Shimadzu LC-10 AD VP with a Shimadzu 3D UV detector and
a Gen-Pak FAX column (Waters, 4.6 × 100 mm); a linear
gradient (10-67%) of solvent III [1 M NaCl in 25 mM
phosphate buffer (pH 6.0)] in solvent IV [25 mM phosphate
buffer (pH 6.0)] was used at 50 °C at a flow rate of 1.0 mL/
min for 40 min. ESI mass was performed by use of Mariner
(PerSeptive Biosystems Inc.). MALDI-TOF mass was performed
using a Bruker Daltonics [Matrix: 3-hydoroxypicolinic acid (100
mg/mL) in H2O-diammonium hydrogen citrate (100 mg/mL)
in H2O (10:1, v/v)].
Synthesis of Compound 3. 6-N-Phenoxyacetyl-5′-O-(4,4′-
dimethoxytrityl)-2′-O-cyanoethyladenosine (1) (0.25 g, 0.33
mmol) and imidazole (0.13 g, 1.83 mmol) in CH2Cl2 (1.65 mL)
were added to the solution of compound 2 (0.37 mmol) in
CH2Cl2 (1.85 mL) under an argon atmosphere. After the mixture
was stirred at room temperature for 90 min, the mixture was
partitioned between CHCl3 and brine. The organic phase was
collected and evaporated under reduced pressure. The residue
was chromatographed on a column of silica gel with CHCl3
containing 1% Et3N to give the fractions containing the desired
silylated nucleoside. The fractions were collected and evaporated
under reduced pressure. The residue was finally evaporated by
repeated coevaporation three times with toluene and CHCl3 to
remove the last traces of Et3N. Subsequently, the residue was
dissolved in dry CH3CN (2.8 mL). 1,8-Diazabicyclo[5.4.0]-7-
undecene (170 µL, 1.13 mmol) was added to the mixture. After
the mixture was stirred at room temperature for 15 min, a 0.2
M solution of triethylamine hydrogen carbonte (1.4 mL) was
added. After being stirred at room temperature for 5 min, the
mixture was partitioned between CHCl3 and brine. The organic
phase was collected and evaporated under reduced pressure. The
residue was chromatographed on a column of silica gel with
Moreover, the 2′-O-methylation also stabilizes hydrated
waters in the minor groove.17 If the water molecules in the minor
groove were stabilized so that they could remain unmoved for
a long time, it could reduce the fluctuation of the base-pair steps.
Thus, the stabilization of the hydration by the 2′-O-methylation
could also contribute to the large Ftotal
.
These explanations also raised the limitation of this method.
First, the modifications that affected the electrostatic repulsion
among phosphate groups, such as modifications with positive
or negative charges, would not be estimated well because the
change of the electrostatic repulsion could increase or decrease
the duplex stability without affecting the deformability. In
addition, since our method was only derived from the calcula-
tions at the duplex states, care should be taken for the
modifications that can drastically change the properties of the
single strand states. BNA or LNA, which has perfectly fixed
sugar moieties even in the single strand, is an example.
Furthermore, the linear functions of the deformability and
Tm values are different among base sequences (Figure 6, Table
S5 (Supporting Information)). This would mean that our method
cannot be used for comparable study between the duplexes of
different base sequences. Therefore, in practice, one must
measure the experimental values of at least two modified or
unmodified duplexes of each targeted base sequence.
It should be also noted that it was also difficult to explain
the deformability from the aspect of entropy or enthalpy
contributions (Figure S6, Supporting Information). These analy-
ses indicated that the more rigid base pair steps did not
necessarily mean the base pair steps of the lower entropy state.

2522 J. Phys. Chem. B, Vol. 114, No. 7, 2010
Masaki et al.
CHCl3-MeOH (100:0-95:5, v/v) containing 1% Et3N. The
fractions were collected and evaporated under reduced pressure.
The residue was finally evaporated by repeated coevaporation
three times with toluene and CHCl3 to give compound 3 (0.25
sodium phosphate, and 0.1 mM EDTA adjusted to pH 7.0. The
solution was kept at 85 °C for 10 min for complete dissociation
of the duplex to single strands, cooled at a rate of 1.0 °C/min
and kept at 15 °C for 10 min. After that, the melting
temperatures (Tm) were determined at 260 nm using a UV
spectrometer (Pharma Spec UV-1700TM, Shimadzu) by increas-
ing the temperature at a rate of 1.0 °C/min.
1
g, 64%). H NMR (CDCl3): δ 0.92 (d, 3H, J ) 7.5 Hz), 0.94
(d, 3H, J ) 8.0 Hz), 1.00 (d, 3H, J ) 7.5 Hz), 1.02 (d, 3H, J
) 8.0 Hz), 1.04-1.16 (m, 1H), 1.28-1.32 (m, 3H), 1.95-2.06
(br s, 2H), 2.50-2.57 (m, 2H), 2.57-2.62 (br s, 2H), 3.23 (d,
1H, J ) 10.0 Hz), 3.49-3.68 (m, 4H), 3.70-3.82 (m, 8H),
4.31-4.38 (br s, 1H), 4.48 (s, 1H), 4.70-4.77 (br s, 1H), 4.81
(s, 1H), 6.18 (s, 1H), 6.72-6.82 (br s, 4H), 7.01-7.10 (m, 3H),
7.15-7.25 (m, 6H), 7.29-7.40 (m, 4H), 7.55-7.61 (m, 3H),
7.78 (d, 2H, J ) 8.0 Hz), 8.17 (s, 1H), 8.74 (s, 1H), 9.46-9.47
(brs, 1H). HRMS (ESI) m/z (M + H) calcd for C59H66N7O11Si+:
1076.4584. Found: 1076.4580.
Computational Methods. The simulations were carried out
using the AMBER 9.0 program package with the parmBSC0
revision of the parm99 Cornell et al. force field.31,32 The initial
structures of each duplex were derived from NUCGEN module
embedded in AMBER. The charge of the 2′-O-methyl group
(2′-OMe) was taken from the Auffinger’s parameter.17 Param-
eterization of the 2′-O-cyanoethyl (2′-OCE) and 2′-O-methoxy-
ethyl (2′-OMOE) groups was done using the RESP/6-31G(d)
charges.33-35 The additional force field parameters of the 2′-
OCE group were taken from the gaff force-field parameters.36
The sequence of calculated 2′-O-modified-RNA/RNA hetero
duplexeswereU14/A14,(UUAA)5UUA/(UAAU)5UAA,(CCGG)5CCG/
(CGGC)5CGG, and (ACUG)5ACU/(AGUC)5AGU. The RNAs
were solvated in a periodic box with a 10 Å buffer of water
molecules, explicitly described by the SPC/E model and
neutralized, resulting in a concentration of added NaCl of
approximately 0.1 M.37 The parameters included in ions94.lib
file embedded in AMBER 9.0 were used for Na+ and Cl- ions.
An initial optimization of 1000 cycles, the first 500 cycles by
steepest descent and the rest with a conjugate gradient method,
was performed with the duplex constrained 500 kcal/(mol ·Å2)
to relax the solvent. Then, a further optimization of 5000 cycles
with no constraints on the whole system was carried out to lead
to a final relaxed geometry. The first equilibrations were carried
out with a 10 kcal/(mol ·Å2) constraint on the duplex for 100
ps at constant volume, constantly increasing the temperature
from 0 to 300 K. Next, the equilibrations were continued to
200 ps at a constant pressure of 1 atm, and the temperature
was kept constant with the Langevin algorithm. The production
simulations were performed for 25 ns (U14/A14) or 20 ns (the
others) with the Berendsen algorithm to maintain the temper-
ature.38 The time constant (1.0 ps) for the heat bath coupling
was used. During the MD calculation, hydrogen vibrations were
removed using SHAKE bond constraints, allowing a longer time
step of 2 fs.39 Long range electrostatic interactions were treated
using the particle mesh Ewald approach and a 10 Å cutoff.40
Harmonic Stiffness Analysis. Harmonic stiffness analysis
for the helical parameters of nucleic acids, such as twist, roll,
tilt, rise, shift and slide, was introduced by Lankas et al. and
Olson et al. to quantitatively analyze the deformability of nucleic
acid duplexes.22-24 The harmonic stiffness analysis is based on
the identical form of the Gauss distribution function (eq 2) and
Einstein’s formula of a harmonic oscillator (eq 3).
Synthesis of Compound 4. A CPG resin (1 g, 20 µmol) was
coevaporated with dry pyridine three times and dried under
reduced pressure. The CPG resin was added in dry CH2Cl2 (10
mL) in a round flask; compound 3 and DCC (0.1 g, 500 µmol)
were added to the mixture. After the round flask containing the
mixture was attached to a rotary evaporator and gently rotated
for 12 h, the solvent was removed by filtration. The residual
CPG was washed by CH2Cl2 and dried under reduced pressure.
The resin was then dissolved in pyridine-Ac2O (9:1, v/v, 30
mL) in a round flask and 4-(dimethylamino)pyridine (80 mg,
640 µmol) was added. The round flask containing the mixture
was attached to a rotary evaporator and gently rotated for 2 h,
and the solvent was removed by filtration. The residual CPG
resin 4 was washed with CH3CN and dried under reduced
pressure.
Synthesis of Oligoribonucleotides. The synthesis of oligori-
bonucleotides UOMOE and UOCE was carried out on a CPG
14
14
resin having a universal support III and a succinyl linker in an
ABI 392 DNA synthesizer, respectively. Then, the UOMOE14 and
UOCE14 oligomers were released from the resin by treatment with
a solution of 2.0 M methanolic ammonium and aqueous
NH3-NH4OAc (10:1, v/w) solution, respectively, at room
temperature for 1 h. The polymer supports were removed by
filtration and washed with 0.1 M ammonium acetate buffer (1
mL × 3). The filtrates were purified by anion-exchange HPLC
to give oligoribonucleotides UOMOE14 and UOCE14. The synthesis
of oligoribonucleotides AOCE14 was carried out on a CPG resin
having a silyl linker in an ABI 392 DNA synthesizer. The fully
protected oligomer, after chain elongation, was deprotected by
treatment with an aqueous NH3-NH4OAc (10:1, v/w) solution
at room temperature for 45 min. Then, the mixture containing
oligoRNAs was released from the resin by treatment with a
solution of Et3N·3HF (0.2 M) and Et3N (0.4 M) in THF (500
µL) at room temperature for 4 h. The polymer support was
removed by filtration and washed with 0.1 M ammonium acetate
buffer (1 mL × 3). The filtrate was purified by anion-exchange
HPLC to give oligoribonucleotides.
(x - µ)2
Oligoribonucleotide: AOCE
MALDI-TOF Mass (M + H) calcd for C183H214N84O81P13
5286.2;. Found: 5291.2.
Oligoribonucleotide: UOCE
MALDI-TOF Mass (M + H) calcd for C168H198N42O110P13
4965.8. Found: 4970.4.
Oligoribonucleotide: UOMOE
MALDI-TOF Mass (M + H) calcd for C168H240N28O124P13
5036.0. Found: 5036.5.
P(x) ) Nexp -
(2)
(3)
14
(
)
2σ2
+
:
:
:
f(x - µ)2
1
14
P(x) ) Nexp -
·
(
)
+
2
kBT
14
where x is any structural parameter, µ is the average of x, σ is
the standard deviation, f is the elastic constant, N is the
normalization constant, kB is Boltzmann’s constant, and T is
the absolute temperature, respectively. In the simple case, by
comparing these two formulas, one can calculate the f value as
(eq 4) from the standard deviation (σ) obtained by analyzing
+
UV Melting Curve Analysis. An appropriate oligoribonucle-
otide (2 µM) and its complementary ssRNA (2 µM) were
dissolved in a buffer consisting of 100 mM NaCl, 10 mM

Deformability of 2′-O-Modified RNA Hetero Duplexes
J. Phys. Chem. B, Vol. 114, No. 7, 2010 2523
the distribution of parameter x throughout the molecular dynamic
simulation.
pairs (omitting terminal 3 base pairs) and averaged same base
pair steps to provide more reliable values.
Acknowledgment. This study was supported in part by a
grant from the Genome Network Project from the Ministry of
Education, Culture, Sports, Science and Technology, Japan, and
by the G-COE project. This study was also supported in part
by a KAKENHI 20350074 from the Ministry of Education,
Culture, Sports, Science, and Technology, Japan, and Research
for Promoting Technological Seeds program of JST.
kBT
σ2
f )
(4)
In complex molecules, such as DNA duplexes, each of the
structural parameters, such as roll, rise, shift, slide, tilt, and twist,
are correlated. Therefore, a covariance-variance matrix (C) of
six parameters (twist, roll, tilt, rise, shift, and slide) was used
instead of the σ2 value.
Supporting Information Available: Detailed RESP charges
and force field parameters of modified moiety, base-pair step
data, rms deviation of each trajectory, and realtionship between
deformability and thermodynamic parameter. This material is
available free of charge via the Internet at http://pubs.acs.org.
F ) kBTC-1
(5)
where F is the stiffness matrix associated with helical deforma-
tion at the base pair step.
A global view of helical deformability can be obtained by
defining the translational (Ftrans), rotational (Frot), and their
product (Fprod) deformability indexes.25
References and Notes
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JP909851J