Probing specific RNA bulge conformations by modified fluorescent nucleosides

Article abstract of DOI:10.1039/b816768k

Recently, RNA bulges, three-dimensional structural motifs that function as molecular handles in RNA, were identified in various sequences and have been proposed as the interaction site for RNA-binding drugs. However direct and sensitive monitoring methods

Full text of DOI:10.1039/b816768k

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Probing specific RNA bulge conformations by modified
fluorescent nucleosides†
Hyun Seok Jeong,a,b Sunwoo Kang,c Jin Yong Lee*c and Byeang Hyean Kim*a
Received 24th September 2008, Accepted 28th November 2008
First published as an Advance Article on the web 23rd January 2009
DOI: 10.1039/b816768k
Recently, RNA bulges, three-dimensional structural motifs that function as molecular handles in RNA,
were identified in various sequences and have been proposed as the interaction site for RNA-binding
drugs. However direct and sensitive monitoring methods specific to RNA bulges have not been well
developed. We describe here pyrene-labeled uridine derivatives which were incorporated into HIV TAR
RNA bulge sites resulting in greatly enhanced fluorescence, allowing the effective monitoring of base
conformations in HIV TAR RNA. To clarify this fluorescence enhancement, Hartree–Fock
calculations were performed for natural and pyrene-labeled HIV TAR RNA. The calculated results
showed a considerable rotation of the pyrene moiety at U25 due to argininamide-induced
conformational changes in the RNA bulge. This simple and efficient method can be used to elucidate
conformational changes in specific bulge regions.
HIV TAR RNA is known to contain specific RNA bulge sites
Introduction
(UCU) that specifically interact with Tat protein, an essential
factor for viral replication.6 This interaction can be reduced to its
components, the HIV TAR RNA bulge and a Tat peptide mimic,
argininamide (ARG).
RNA bulges are common and recurring motifs in three-
dimensional structures in RNA. They consist of unpaired bases
typically located between stacked base pairs in duplex RNA
to create unique recognition sites.1 Recently, RNA bulges were
discovered in various RNA structures such as HIV trans-activator
responsive (TAR) RNA, RNA splicing intermediates, and 5S
ribosomal RNA.2 Bulge structures are highly conserved and
function as molecular handles or specific binding sites by widening
or compressing RNA grooves. Consequently, RNA bulges can be
a spotlighting target of RNA binding drugs.3
To monitor these conformational changes, we incorporated a
fluorescent nucleoside developed by our research group8 and other
groups9 into an RNA nucleoside to yield a novel pyrene-labeled
uridine nucleoside. Covalently linked ethynyl pyrene gives several
important advantages. First, the ethynyl moiety at uridine C-5 is
presumably a strong and stable bond that is not likely to perturb
RNA base pairing or duplex stability.10 Second, the fluorophore
pyrene is well characterized and sensitive to the hydrophobicity of
its surrounding environment. Since the RNA bulge conformation
depends on interactions with other stacked bases, the covalently
bound ethynyl pyrene can be an effective means of probing RNA
bulge residues.
RNA bulge conformation may be stacked or unpaired, orien-
tated into the groove or as flap residues.4 Therefore to quickly
characterize a bulge and its interactions, it is important to
develop effective and simple means of monitoring RNA bulge
conformations. Although several approaches have emerged,5
a
direct and sensitive method is lacking. In this study, a new method
for monitoring RNA bulge conformation was developed using a
fluorescent nucleoside based on reasonable ab initio Hartree–Fock
(HF) calculations.
In the free HIV TAR RNA, bulge residues at U23 RNA base
and U25 RNA base are stacked in the RNA groove, but when
argininamide binds to RNA, both U23 RNA base and U25
RNA base undergo considerable changes due to ligand–RNA
interactions.7
Results and discussion
First, pyrene-labeled uridine phosphoramidite 5 was synthe-
sized from 5-iodo-5¢-dimethoxytrityl-2¢-tert-butyldimethylsilyl-
dimethyluridine 3 through a palladium-catalyzed Sonogashira
coupling intermediate 4 (Fig. 1). The corresponding phospho-
ramidite was incorporated into HIV TAR RNA by automated
RNA synthesis.
Three possible uridine RNA bulges can occur in HIV TAR
RNA at U23, U25, and U31 (Fig 2a). As a control, an unmodified
HIV TAR RNA 6 was synthesized in addition to the three RNA
(U23, U25, U31) 7–9 including pyrene-labeled uridine (Fig. 2b) in
site specific manners (Fig. 2c).
The fluorescence emission of the pyrene-labeled RNA bulge
increased with the concentration of argininamide (Fig. 3a). The
overall shape of the fluorescence spectra varied among the pyrene-
labeled RNA bulges due to different stacking arrangements with
the RNA bases. Specifically, the fluorescence intensity of pyrene-
labeled U25-RNA 8 increased approximately 6.2 fold with the
aDepartment of Chemistry, BK School of Molecular Science, Pohang, 790-
784, Korea
bSchool of Interdisciplinary Bioscience and Bioengineering (I-BIO), Pohang
University of Science and Technology, Pohang, 790-784, Korea. E-mail:
bhkim@postech.ac.kr
cDepartment of Chemistry, Institute of Basic Science, Sungkyunkwan
University, Suwon, 440-746, Korea
† Electronic supplementary information (ESI) available: The initial struc-
tures for each modeling complex, 1H NMR spectra for compound 2–5,
and MALDI-TOF data and MALDI-TOF spectra for synthesized RNA
6–9. See DOI: 10.1039/b816768k
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Fig. 1 Synthesis of pyrene labeled uridine phosphoramidite: (i) DMTrCl,
DMAP, pyridine, rt, 3 h, 84%; (ii) TBDMSCl, AgNO3, pyridine, THF,
rt, 2 h, 66%; (iii) 1-ethynylpyrene, (PPh3)4Pd, CuI, DIPEA, DMF,
45–50 ◦C, 2 h, 94%; (iv) 2-cyanoethyldiisopropylchlorophosphoramidite,
4-methylmorpholine, CH2Cl2, 0 ◦C, 5 h, 85%.
Fig. 3 (a) Fluorescence spectrum of U23, U25, and U31 RNA (7–9)
in or without 3.2 mM of argininamide (ARG). (b) Graph of fluorescent
enhancement of U23, U25, U31 RNA (7–9). All of the data in (a) and (b)
were recorded at 1 mM RNA concentration.
Fig. 2 HIV TAR RNA structure and pyrene-labeled RNA: (a) the
sequences of the HIV TAR RNA element; (b) the structure of pyrene
labeled uridine monomer; (c) the sequences of natural RNA 6 and pyrene
labeled RNA 7–9.
addition of 3.2 mM argininamide. In contrast, the fluorescence
emission of pyrene from labeled U23-RNA 7 and U31-RNA
9 bulges increased only by factors of 1.4 and 1.6, respectively,
at the same argininamide concentration (Fig. 3b). The total
discriminating factors were 14.1 and 8.7 between U25-RNA 8
and U23-RNA 7 and between U25-RNA 8 and U31-RNA 9,
respectively.
Duplex melting temperatures (Tm) were recorded for both
natural and pyrene-labeled RNA to confirm that the pyrene
moiety did not interrupt the stability of the HIV TAR RNA
complex. Thermal denaturation studies indicated that pyrene-
labeling resulted in a slight enhancement relative to natural RNA
(Fig. 4). This result also provides strong evidence of the close
interaction of pyrene-labeled uridine base into the other RNA
bases.
Circular dichroism spectra exhibited specific band changes
at 230 nm after the addition of argininamide (Fig. 5). These
bands were identical in both natural and pyrene-labeled HIV
TAR RNA. Duplex melting temperature (Tm) data and circular
dichroism data showed that pyrene labeling of RNA has little
perturbation effect on natural RNA.
Fig. 4 Melting temperature spectra of natural and modified oligonu-
cleotides (ODNs). The spectra were recorded at 20 ◦C in a buffer of
100 mM Tris–HCl, 100 mM NaCl (pH 7.2). Each ODN concentration
was 1 mM and the absorption wavelength was 260 nm.
RNA upon binding to argininamide (ARG), Hartree–Fock (HF)
calculations were performed for natural RNA 6, UPY23-RNA 7,
UPY25-RNA 8, natural RNA 6 with argininamide, UPY23-RNA 7
with argininamide and U25-RNA 8 with argininamide complexes
To understand the modification effect of pyrene labeling more
exactly and the fluorescence enhancement of U23-RNA and U25-
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in bulk and not in molecule–molecule interactions cannot be
considered even in the current ab initio calculations based on self-
consistent reaction field (SCRF) methods because the main origin
of the hydrophobic interaction is the entropic contribution by
forming a huge cavity in solvent water, which can not be considered
in current ab initio calculations. The pyrene–RNA interactions
calculated in the gas phase may change in water solution, which
gives the enthalpic contribution. In our previous studies on enzyme
reactions, the enzyme–substrate interaction was often electrostatic
(dipole–dipole interaction) in the hydrophobic pocket (active sites)
and the gas phase results gave reliable results. This electrostatic
interaction in a hydrophobic pocket is the reverse of the current
case of hydrophobic interactions in water solution, thus our
calculations in the gas phase can give reasonable conclusions
even though the pyrene–RNA interaction may change in water
solution.
The modification effect of pyrene labeling at each bulge was
estimated by the rotation angles at U23 and U25 bases. The
changes in the dihedral angle between natural RNA and pyrene
modified RNA were about 15◦ at U23 (57.2◦ in natural RNA
6, 42.5◦ in UPY23-RNA 7) and U25 (75.4◦ in natural RNA 6,
62.2◦ in UPY25-RNA 8). These values showed that pyrene labeling
of an RNA base did not change the RNA base orientations
and maintained the original RNA base orientations. The next
calculated results indicated that the binding of argininamide
induced a major shift in the rotational position of pyrene and
a net movement of the pyrene moiety away from the RNA base
pair backbone. The change in the dihedral angle of pyrene in
UPY25-RNA upon argininamide binding (73.1◦) was much larger
than the corresponding change for UPY23-RNA (15.3◦). These
results clearly support the observed fluorescence enhancement of
UPY25-RNA upon argininamide binding. In addition, the change
in the dihedral angle of natural RNA showed similar changes
in the uridine base at U23 (18.2◦) and U25 (61.5◦) compared to
pyrene labeled RNA. Therefore pyrene labeling has little effect on
RNA local structures, it does not change much the rotation angles
upon argininamide binding. The initial and optimized structures
for each structures are shown in the ESI.†
Fig. 5 Circular dichroism spectra of natural (green) and modified ODNs
(orange). The spectra were recorded at 20 ◦C in a buffer of 100 mM
Tris–HCl, 100 mM NaCl (pH 7.2) with or without 3.2 mM of argininamide
(ARG). Each natural and modified ODNs concentration was 1 mM.
using a STO-3G basis set in Gaussian 03 software11 (Fig. 6) in the
gas phase. Due to limited computational capabilities, a simplified
model composed of seven base pairs and one bulge site was
employed to mimic entire HIV TAR RNA to precisely simulate
its interaction with argininamide. Several argininamide units
can bind with HIV TAR RNA, however the specific argini-
namide which binds to the specific RNA bulge site was mainly
considered.
In HIV TAR RNA without argininamide, fluorescence from
pyrene-labeled RNA uridine is quenched by interactions with
adjacent base pairs through photo-induced electron transfer
(PET).12 Since RNA bulge sites are typically intercalated in
base pair stacks, ligands or proteins that bind at the bulge site
can decrease base pair stacking, thereby exposing buried RNA
residues. When argininamide binds with HIV TAR RNA, the U25
base is reoriented outside of the RNA groove, diminishing base
pair quenching effects and resulting in increased fluorescence with
a strongly emissive pyrene.
The fluorescence of the covalently linked ethynyl pyrene de-
scribed herein is sensitive to these changes in base interaction
and can be a useful tool to measure RNA bulge conformation.
This technique provides several advantages. First, base pair
orientational changes can be measured, since conformational
changes in RNA bulges depend on base pair orientation. Second,
the environment surrounding the uridine base to which the ethynyl
pyrene moiety is attached is probed directly. Similar results were
obtained using 2¢-amino-butyryl-pyrene uridine with the pyrene
moiety attached at the 2¢-hydroxyl group of sugar ring.13 Third,
relative to NMR spectroscopy, the use of a labeled nucleoside
Fig. 6 The optimized structures of natural RNA, natural RNA-ARG,
UPY23-RNA, UPY25-RNA, UPY23-RNA-ARG, and UPY25-RNA-ARG
providing dihedral angle values.
In addition, the pyrene moiety is hydrophobic and the water–
pyrene interaction should be small. The pyrene–RNA interactions
in water solution may be different from the pyrene–RNA interac-
tions in the gas phase. However, such hydrophobic interactions
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represents a much simpler and more efficient probing method.
Fourth, compared to other monitoring methods,14 the sensitivity
of pyrene-labeled uridine affords detection of minute structural,
conformational, and orientational changes in RNA bulges.
CDCl3): d 8.20 (s, 1H; H6), 7.24–7.41 (m, 9H; DMT), 6.85-6.82
(d, J = 8.7 Hz, 4H: DMT), 5.98 (s, 1H; H1) 4.48 (t, J = 4.80 Hz,
1H; H2), 4.24 (q, 1H, J = 3.94 Hz; H3) 4.16 (br s, 1H; H4), 3.78 (s,
6H, OCH3), 3.44 (d, J = 9.2 Hz, 2H; H5¢), 3.38 (d, 2H, J = 9.2 Hz,
H5¢¢). 0.91 (s, 9H, t-butyl). 13C NMR (75 MHz, CDCl3): d 158.2,
149.3, 143.8, 134.8, 134.7, 129.6, 127.7, 127.5, 126.7, 112.9, 87.6,
86.8, 83.6, 71.0, 62.7, 54.9, 25.1, 17.5. FAB (m/z): [M + Na]+ calcd
for C36H43IN2O8SiNa, 809.17; found, 809.05.
Conclusions
In conclusion, a pyrene-labeled uridine derivative was synthesized
by Sonogashira coupling to probe base pair conformation and
stacking interactions in RNA bulges. The fluorescence discrimi-
nating factors of the pyrene moiety were so high that it is sufficient
to detect changes in RNA base conformations, for example, in
HIV TAR RNA with argininamide. Pyrene labeling of RNA has
a minimum effect on RNA natural structures and the modeling
results of pyrene labeled RNA upon argininamide binding were
consistent with natural RNA modeling results upon the same
case. Therefore this monitoring method can be used to detect
and characterize conformational changes in RNA or DNA bulge
regions due to targeted binding events such as RNA binding
protein or drug interactions.
5¢-O-[Bis(4-methoxyphenyl)phenylmethyl]-2¢-O-(tert-butyl-
dimethylsilyl-5-(2-pyrenylethynyl)uridine (4)
(PPh3)4Pd (18 mg, 0.015 mmol) and CuI (6 mg, 0.031 mmol)
were added to a solution of 3 (240 mg, 0.305 mmol) in THF
under Ar and then the mixture was stirred for 5 min at room
temperature. 1-Ethynylpyrene (83 mg, 0.366 mmol) was added to
this solution. After degassing, the reaction mixture was stirred at
45 ◦C for 5 h and monitored by TLC. Water was added to the
solution and the product was extracted with excess ethyl acetate.
The organic phase was washed twice with water, After evaporation
of solvent under reduced pressure, the residue was subjected to
column chromatography (SiO2; hexane–ethyl acetate, 2 : 1) to yield
product 3 (254 mg, 94%). [a]2D0 = +16 (CHCl3). IR (film): n 3460,
3200, 3033, 2929, 2856, 1717, 1616, 1506, 1457, 1252, 1218, 848,
Experimental section
5¢-O-[Bis(4-methoxyphenyl)phenylmethyl]-5-iodouridine (2)
1
772 cm-1. H NMR (300 MHz, CDCl3): d 8.41 (d, J = 9.2 Hz,
4,4¢-Dimethoxytrityl chloride (704 mg, 2.08 mmol) was added
to a solution of (-)-5-iodouridine (700 mg, 1.89 mmol) and
4-dimethylaminopyridine (26 mg, 0.21 mmol) in pyridine (10 mL).
After stirring at room temperature for 3 h, the mixture was
concentrated under reduced pressure. A 5% NaHCO3 solution
(10 mL) was added to the mixture, and the solution was extracted
with ethyl acetate 2 times (10 mL), and then dried with sodium
sulfate. Column chromatography (SiO2; hexane–ethyl acetate,
from 1 : 1 for eliminating pyridine, to 1 : 4) gave product 2 as
a white solid in 83% yield. [a]2D0 = +12 (CHCl3). IR (film): n 3400,
3065, 2930, 2835, 1699, 1607, 1508, 1445, 1301, 1250, 1220, 1175,
1095, 1054, 1033, 828, 771, 723, 700, 667 cm-1. 1H NMR (300 MHz,
CDCl3): d 8.23 (s, 1H; H6), 7.19–7.39 (m, 9H; DMT), 6.84–6.80
(d, J = 8.7 Hz, 4H: DMT), 5.86 (s, 1H; H1) 4.46 (t, J = 4.80 Hz,
1H; H2), 4.36 (q, 1H, J = 3.94 Hz; H3) 4.24 (br s, 1H; H4), 3.75
(s, 6H, OCH3), 3.42 (d, J = 9.2 Hz, 2H; H5¢), 3.36 (d, 2H, J =
9.2 Hz, H5¢¢). 13C NMR (75 MHz, CDCl3): d 160.1, 158.2, 150.6,
143.9, 135.0, 134.8, 129.6, 127.7, 127.5, 126.6, 112.9, 90.1, 86.5,
84.2, 75.1, 70.4, 68.5, 66.3, 62.4 54.8. FAB (m/z): [M + Na]+ calcd
for C30H29IN2O8Na, 695.09; found, 695.09.
1H; ArPyH), 8.30 (s, 1H; H6), 8.14 (t, J = 6.8 Hz, 2H; ArPyH), 8.03
(d, J = 8.0 Hz, 1H, ArPyH), 7.98 (d, J = 8.3, 5.4 Hz, 2H; ArPyH),
7.89 (t, J = 8.0 Hz, 2H; ArPyH) 7.56 (d, J = 8.0 Hz, 1H; ArPyH)
7.41–7.24 (m, 9H; DMT), 6.68–6.64 (d, J = 8.7 Hz, 4H: DMT),
6.09 (s, 1H; H1), 4.41 (t, J = 4.80 Hz, 1H; H2), 4.11 (br s, 1H; H4),
4.09 (q, 1H, J = 3.94 Hz; H3), 3.63 (d, J = 9.2 Hz, 2H; H5¢), 3.49
(s, 3H, OCH3), 3.43 (s, 3H, OCH3), 3.40 (d, 2H, J = 9.2 Hz, H5¢¢).
0.93 (s, 9H, t-butyl). 13C NMR (75 MHz, CDCl3):d 160.1, 158.1,
148.8. 144.0, 134.9, 130.8, 131.0, 130.0, 129.9, 129.6, 128.0, 127.7,
127.4, 126.7, 125.6, 125.1, 125.0, 123.7, 125.5, 112.9, 101.3, 87.8,
87.0, 83.4, 71.1, 62.7, 60.0, 54.5, 25.1, 17.6. FAB (m/z): [M + Na]+
calcd for C54H52N2O8SiNa, 908.44; found, 908.44.
5¢-O-[Bis(4-methoxyphenyl)phenylmethyl]-2¢-O-(tert-butyl-
dimethylsilyl-5-(2-pyrenylethynyl)uridine 3¢-O-[2-
cyanoethyl(N,N-diisopropylamino)phosphoramidite (5)
4-Methylmorpholine (74 mL, 0.678 mmol) was added to a solution
of product 3 (100 mg, 9.113 mmol) in CH2Cl2 (5 mL) under
nitrogen and then the mixture was stirred at room temperature for
30 min. 2-Cyanoethyldiisopropyl-aminochlorophosphoramidite
(76 mL, 0.339 mmol) was added and the mixture was stirred
for 3 h and monitored by TLC. The solvent was washed with
5% NaHCO3 and the residue was purified by chromatography on
short column (SiO2; hexane–EtOAc–pyridine, 99 : 99 : 2) to yield
product 4 (85%). [a]1D4 = +15 (CHCl3). IR (film): n 3177, 3015,
2970, 2950, 1733, 1616, 1507, 1456, 1365, 1294, 1217, 1179, 1093,
1034, 846, 772 cm-1. 1H NMR (300 MHz, CDCl3): d 8.41 (s, 1H;
H6), 8.38–8.35 (d, J = 9.2 Hz, 1H; ArPyH), 8.14 (t, J = 6.8 Hz, 2H;
ArPyH), 8.03 (d, J = 8.0 Hz, 1H, ArPyH), 8.00 (d, J = 8.3, 5.4 Hz,
2H; ArPyH), 7.86 (t, J = 8.0 Hz, 2H; ArPyH) 7.67 (d, J = 8.0 Hz,
1H; ArPyH) 7.50–7.24 (m, 9H; DMT), 6.74–6.68 (d, J = 8.7 Hz,
4H: DMT), 6.10 (s, 1H; H1), 4.26 (t, J = 4.80 Hz, 1H; H2), 4.21
(br s, 1H; H4), 4.09 (q, 1H, J = 3.94 Hz; H3), 3.88–3.45 (m, 5H,
5¢-O-[Bis(4-methoxyphenyl)phenylmethyl]-2¢-O-(tert-butyl-
dimethylsilyl)-(-)-5-iodouridine (3)
Dry pyridine (0.36 mL, 4.76 mmol) was added to a solution
of product 2 (600 mg, 0.892 mmol) and silver nitrate (182 mg,
1.07 mmol) in dry THF (10 mL). The reaction mixture was stirred
at room temperature for 15 min, followed by the addition of
tert-butyldimethylsilyl chloride (175 mg, 1.16 mmol). Stirring for
an additional 2 h, the solution was extracted with 5% NaHCO3
solution and ethyl acetate and dried with sodium sulfate. Column
chromatography (SiO2; hexane–ethyl acetate, 2 : 1) gave product
3 as a white solid in 66% yield. [a]2D0 = +16 (CHCl3). IR (film):
n 3464, 3064, 2929, 2956, 1700, 1609, 1507, 1456, 1251, 1219,
1177, 1135, 1091, 1034, 837, 772, 668 cm-1. 1H NMR (300 MHz,
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NCH, OCH2, H5¢), 3.49 (s, 3H, OCH3), 3.43 (s, 3H, OCH3), 3.40 (d,
2H, J = 9.2 Hz, H5¢¢). 2.71 (m, 2H, CH2CN), 1.24–1.15 (2d, 12H,
J = 6.8 Hz; NCHCH3), 0.93 (s, 9H, t-butyl). 13C NMR (75 MHz,
CDCl3): d 160.1, 158.2, 149.3, 144.0, 141.2, 143.8, 134.8, 134.7,
129.6, 127.7, 127.5, 126.7, 112.9, 100.6, 87.6, 86.8, 83.6, 71.0, 62.7,
54.9, 42.8, 30.5, 25.1, 19.7, 17.5. 31P NMR (121 MHz, CDCl3):d
152.1, 150.3. FAB (m/z): [M + Na]+ calcd for C36H43IN2O8SiNa,
809.17; found, 809.05.
Acknowledgements
BHK is grateful to KOSEF for financial support through CRI-
Accelerlation Research grant by MEST (No. R21-2008-035-0100-
0). JYL acknowledges the KOSEF grant by MEST (No. R11-
2007-012-03002-0).
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RNA oligonucleotide synthesis and characterization
To synthesize modified RNA oligonucleotides, we prepared the
DMTr-protected 2-cyanoethyl phosphoramidite derivative and
applied it directly into solid phase oligonucleotide synthesis
protocols using an automated DNA synthesizer, Perceptive
Biosystems 8909 ExpediteTM. The synthesized oligonucleotides
were cleaved from the solid support upon treatment with MeNH2
in H2O–EtOH = 3 : 1 for 5 h at room temperature. The
crude products were lyophilized and diluted with 1-methyl-2-
pyrrolidone–triethylamine–TEA–HF = 6 : 3 : 4 to remove the
protecting 2¢-TBDMS group. After heating the solution for 2 h
at 65 ◦C, the solution was neutralized with TBE solution. The
oligonucleotides were purified using HPLC (Hiber Pre-packed
Column RT 250–10, customized packing Lichrospher 100 RP-
18). The HPLC mobile phase was the gradient between 5%
acetonitrile–0.1 M triethylammonium acetate (TEAA) (pH 7.0)
and 50% acetonitrile–0.1 M TEAA at 2.5 mL min-1. The fractions
containing oligonucleotides were checked by UV and collected.
The collected solution was treated by 1 M NaOAc–AcOH solution
to remove the 5¢-DMTr group. The purified oligonucleotides
were precipitated with 3 M NaOAc–AcOH solution and excess
EtOH. Final products were obtained by desalting of the solution
using an NAP-10 Column (Amersham Bioscience). For char-
acterization of oligonucleotides, matrix-assisted laser desorption
ionization time-of-flight (MALDI-TOF) mass spectroscopy was
used.
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The dihedral angle values of natural RNA, natural RNA-ARG,
UPY23-RNA, UPY25-RNA, UPY23-RNA-ARG, and UPY25-RNA-
ARG were obtained from optimized structures using a dot product
equation, A·B = |AꢀB|cos q. Then, each angle was obtained by
q = cos-1 (A ¥ B/|AꢀB|), where vectors A and B are given in the
black lines in Fig 6.
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The Royal Society of Chemistry 2009
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