Synthesis and characterization of modified nucleotides in the 970 hairpin loop of Escherichia coli 16S ribosomal RNA

Article abstract of DOI:10.1016/j.bmc.2009.07.008

The synthesis of the 6-O-DPC-2-N-methylguanosine (m²G) nucleoside and the corresponding 5′-O-DMT-2′-O-TOM-protected 6-O-DPC-2-N-methylguanosine phosphoramidite is reported [DPC, diphenyl carbamoyl; DMT, 4,4′-dimethoxytrityl; TOM, [(triisopropylsilyl)oxy]methyl]. The availability of the phosphoramidite allows for syntheses of hairpin RNAs with site-selective incorporation of 2-N-methylguanosine modification. Four 18-nt hairpin RNA analogues representing the 970-loop region (helix 31 or h31; U960-A975) of Escherichia coli 16S rRNA were synthesized with and without modifications in the loop region. Subsequently, stabilities and conformations of the singly and doubly modified RNAs were examined and compared with the corresponding unmodified RNA. Thermodynamic parameters and circular dichroism spectra are presented for the four helix 31 RNA analogues. Surprisingly, methylations in the loop region of helix 31 slightly destabilize the hairpin, which may have subtle effects on ribosome function. The hairpin construct is suitable for future ligand-binding experiments.

Full text of DOI:10.1016/j.bmc.2009.07.008

Bioorganic & Medicinal Chemistry 17 (2009) 5887–5893
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and characterization of modified nucleotides in the 970 hairpin
loop of Escherichia coli 16S ribosomal RNA
*
N. Dinuka Abeydeera, Christine S. Chow
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA
a r t i c l e i n f o
a b s t r a c t
The synthesis of the 6-O-DPC-2-N-methylguanosine (m2G) nucleoside and the corresponding 50-O-DMT-
20-O-TOM-protected 6-O-DPC-2-N-methylguanosine phosphoramidite is reported [DPC, diphenyl
carbamoyl; DMT, 4,40-dimethoxytrityl; TOM, [(triisopropylsilyl)oxy]methyl]. The availability of the phos-
phoramidite allows for syntheses of hairpin RNAs with site-selective incorporation of 2-N-methylguano-
sine modification. Four 18-nt hairpin RNA analogues representing the 970-loop region (helix 31 or h31;
U960–A975) of Escherichia coli 16S rRNA were synthesized with and without modifications in the loop
region. Subsequently, stabilities and conformations of the singly and doubly modified RNAs were exam-
ined and compared with the corresponding unmodified RNA. Thermodynamic parameters and circular
dichroism spectra are presented for the four helix 31 RNA analogues. Surprisingly, methylations in the
loop region of helix 31 slightly destabilize the hairpin, which may have subtle effects on ribosome func-
tion. The hairpin construct is suitable for future ligand-binding experiments.
Article history:
Received 21 April 2009
Revised 29 June 2009
Accepted 3 July 2009
Available online 9 July 2009
Keywords:
Helix 31
2-N-Methylguanosine
5-Methylcytidine
Ribosomal RNA modifications
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
5-methylcytidine at position 967 (m5C967) (Fig. 1). Among the
nucleotides present in h31, residue 965 is the least conserved, with
An intriguing question about the structure and function of the
ribosome is the role played by the naturally occurring modified
nucleotides.1–3 The range of modifications includes methylation
on both the nucleoside base and 20-hydroxyl group of the ribose
moiety, pseudouridylation (uridine isomerization), as well as more
elaborate alterations.4 Although the rRNA modifications have only
been mapped for a small number of organisms, it appears that
most, if not all, ribosomes contain modified nucleotides. It has been
suggested that the modified nucleotides of the small subunit have
important roles in ribosome assembly and protein synthesis, de-
spite the fact that unmodified ribosomes are able to carry out
key biological functions in vitro .5–8 The Escherichia coli (E. coli)
small subunit RNA (16S rRNA) contains one pseudouridine and 10
methylated nucleotides (http://library.med.utah.edu/RNAmods);
m7G527, m2G966, m5C967, m2G1207, m4Cm1402, m5C1407,
m3U1498, m2G1516, m26A1518, and m26A1519.9–11 In 3D models
of the 30S subunit, based on high-resolution X-ray crystal struc-
tures, 8 of the 10 methylated residues cluster in the subunit cleft
near the decoding region.3,12,13
a U present in 52% of known sequences, and A (37%), C (9%) or G
(2%) in the remaining sequences.18 In contrast, nucleotide 966 is
the most conserved, with G occurring in 97.5% of the sequences.18
Similarly, nucleotide 967 is a C in 85% of the sequences and an A in
13%.18 Mutations at either G966 or C967, as well as loss of methyl-
ation at G966, do not affect the growth rate of E. coli;19,20 however,
a single-base deletion at C967 leads to a dominant lethal pheno-
type.19 Recently, a saturation mutagenesis study on the 970 loop
revealed that mutations at positions 966 and 969 significantly af-
fect protein synthesis by the ribosome.21 It was also shown that
ribosomes with single mutations at positions m2G966 and
m5C967 are capable of producing more protein than the wild-type
ribosome.21 These studies suggest that modifications in the 970
loop influence the function of the ribosomal machinery.
There is a considerable amount of structural information for
h31 from ribosome X-ray crystal structures.12,15–17,22,23 In E. coli
70S ribosomes, a three-base stacking interaction is observed
between residues 966, 967, and 968.23 In the crystal structure of
Thermus thermophilus 30S ribosomes, stacking is observed between
those same residues, as well as between positions 965, 969, and
970.22 In addition, the stacked arrangement formed by residues
965, 969, and 970 shows interactions with the ribosomal protein
S13.22 Residue G971 makes six hydrogen bonds within a pocket
formed by residues 949, 950, 1363, 1364, and 1365 in the 16S
rRNA.22 In the ribosome, m2G966 is also protected by the P-site-
bound tRNA from chemical probes.24 Direct contacts between
The 970 loop (helix 31, h31) of E. coli 16S rRNA is located near
the ribosomal P site and therefore believed to be intimately
involved in translation.14–17 E. coli h31 contains two modified
nucleotides, N2-methylguanosine at position 966 (m2G966) and
* Corresponding author. Tel.: +1 313 577 2594; fax: +1 313 577 8822.
E-mail address: csc@chem.wayne.edu (C.S. Chow).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.008

5888
N. D. Abeydeera, C. S. Chow / Bioorg. Med. Chem. 17 (2009) 5887–5893
Figure 1. The structures of N2-methylguanosine (m2G) and 5-methylcytidine (m5C) are shown. Secondary structure representations of E. coli helix 31 of the small subunit
ribosomal RNA with indications of the modification sites and the synthetic RNA hairpins derived from residues 960–975 are shown (unmodified (ECh31UNMOD), fully
modified (wild-type or ECh31WT, containing m2G966 and m5C967), m5C967 singly modified (ECh31M5C), and m2G966 singly modified (ECh31M2G)). The terminal G–C base
pair (g1–c18) was added to stabilize the short stem region.
residues of the tRNA anticodon loop, the P site, and h31 of 16S
rRNA were observed in X-ray crystal structures of the 70S ribo-
some.15,16 These structures suggest that the anticodon loop of the
P-site tRNA is held in place by two stacking interactions; one be-
tween m5C1400 of 16S rRNA and G34 of the tRNA, and a second be-
tween m2G966 of h31 and ribose 34 of tRNA.16,25 The m22G966
residue is flipped out in the 70S ribosomal complex of T. thermophi-
lus containing a model mRNA and two tRNAs.16 In the E. coli 70S
ribosome crystal structure with a tRNA analogue and mRNA, the
corresponding m2G966 residue resides in a tight binding pocket
with the anticodon and the decoding site, which differs from the
30S structure.22 These data suggest that the 970 loop is dynamic
and may be involved in proper positioning of tRNA in the ribo-
somal P site. Modeling studies by Aduri et al.13 suggested that
methylation of residues 966 and 967 increases the surface area
for stacking and also improves van der Waals contacts with the
hydrophobic portion of Arg128 of the S9 protein in the ribosome.
We were interested in developing a better understanding of the
structural and possible stabilizing roles of the modifications in h31
of 16S rRNA. Currently, information is limited regarding the effects
of nucleotide modifications on rRNA structure and function. Until
recently, the chemical properties of the modified nucleotides had
not yet been found to influence specific functional roles in the ribo-
some.26 Nevertheless, even minor changes in chemical composi-
tion of the RNA nucleotides can lead to altered steric properties,
hydrogen-bonding interactions, local base-stacking potential, van
der Waals interactions, or structural rigidity.2 The goals of this re-
search were to synthesize N2-methylguanosine and its correspond-
ing phosphoramidite, and then utilize the amidite in the syntheses
of h31 model sequences. The syntheses were then followed by bio-
physical studies in order to understand the influence of base
methylations on structure and stability of h31 in a systematic man-
ner. The structures and stabilities of four model h31 RNAs were
examined by using circular dichroism (CD) and thermal melting
studies.
2. Results and discussion
2.1. Synthesis of N2-methylguanosine and the corresponding
phosphoramidite
The methylated guanosine nucleoside, m2G, was synthesized by
using a combination of several published procedures (Scheme 1).
The first step involved the acetylation of guanosine to give com-
pound 1.27 The second and third steps to generate N-methylated
intermediate 3 were adapted from Bridson and Reese’s method
using a p-thiocresol intermediate.28 The generation of compounds
2 and 3 was accomplished with 75% yields for each step. To avoid
solubility problems typically encountered during the phospho-
ramidite synthesis of guanosine derivatives, we used a strategy
that involves protection at the lactam function of the guanine res-
idue according to a procedure devised by Kamimura et al.29 and
used by others.30 Diphenylcarbamoyl (DPC) protection and acetyl
deprotection under mild basic conditions gave compound 5 in
good yield (55%, two steps). In addition to the successful O6-protec-
tion of the guanine residue, the presence of DPC improved the sol-
ubility and chromatographic purification properties of the
resulting derivative. The corresponding phosphoramidite was gen-
erated by using a similar approach as Höbartner and coworkers,31
but employing the DPC-protected precursor (compound 5). In our
hands, compound 5 gave higher yields in the m2G phosphorami-
dite (compound 8) synthesis (Supplementary data) than the meth-
ods reported previously.31,32 The synthesis could be performed on
a reasonably high scale to produce sufficient amounts of amidite 8
for multiple couplings.

N. D. Abeydeera, C. S. Chow / Bioorg. Med. Chem. 17 (2009) 5887–5893
5889
Scheme 1. Synthesis of the 6-O-DPC-2-N-methylguanosine 5: (i) acetic anhydride, DMAP, Et3N, acetonitrile, 0.5 h; (ii) p-thiocresol, acetic acid, formaldehyde, ethanol, reflux;
(iii) NaBH4, DMSO, 100 °C; (iv) DPCCl, diisopropylethylamine, pyridine; (v) (a) 2 M NaOH, 20 min; (b) acetic acid.
2.2. Synthesis and characterization of RNA analogues
RNAs is ECh31UNMOD > ECh31M2G > ECh31M5C P ECh31WT.
The RNA samples were analyzed as five different dilutions per
experiment and UV melts were also performed in triplicate for
each construct. Experiments in K+ buffer (15 mM KCl, 20 mM cac-
odylic acid, 20 mM Tris [basic form], 0.5 mM Na2EDTA, pH 7.0)
gave similar results (Supplementary data).
The subtle destabilizing effect of the methylations was some-
what unexpected. In X-ray crystal structures of E. coli 70S ribo-
somes23 and T. thermophilus 30S ribosomes,22 m2G and m5C are
involved in a three-base stacking interaction with residue 968.
One might expect the methyl groups in the modified nucleosides
to facilitate stacking interactions;13 however, both m2G and m5C
are destabilizing relative to standard nucleotides G and C within
the given sequence context. The presence of the h31 methylations
might serve another purpose, such as stabilizing the ribosome
through hydrophobic interactions with Arg128 of the S9 protein.13
Furthermore, the slight destabilization caused by the base methyl-
ation may facilitate base flipping of m2G966, which has been ob-
served in ribosome crystal structures and likely plays an
important role in protein synthesis.16 The small energetic penalty
Fig. 1 shows the RNA analogues representing the 970 stem-loop
region (h31) of E. coli 16S rRNA. The numbering system is based on
the full-length E. coli rRNA sequence. A terminal G–C base pair was
added to each stem (denoted by lower case g–c) in order to stabi-
lize the hairpin structure. Residues in all four constructs are num-
bered from g1 to c18 for the ends and U960 through A975 for the
component representing the natural E. coli h31 sequence. Two
h31 RNAs (ECh31UNMOD and ECh31M5C) were obtained using
commercial amidites. The two remaining h31 analogues
(ECh31M2G and ECh31WT) were synthesized using the 50-O-
DMT-20-O-TOM-6-O-DPC-2-N-methylguanosine phosphoramidite
(compound 8) along with the commercially available amidites.
The doubly modified h31 analogue (ECh31WT) represents the
wild-type sequence of E. coli 16S rRNA helix 31. The incorporation
of m2G and m5C residues was confirmed by MALDI-TOF mass spec-
trometric analysis of purified full-length RNA and by P1 nuclease
digestion and calf intestinal phosphatase treatment of the RNAs,
followed by reverse-phase HPLC analysis of the enzyme digest
products (see Supplementary data).
(DDG°37 = 0.5 kcal/mol) would then be overcome by contacts with
various ribosome components such as 16S rRNA helices, ribosomal
proteins, and tRNA.16 Although the effects on stability may appear
to be quite small, this level of destabilization could account for an
approximate twofold difference in binding to a ribosomal RNA
component, which may be important for fidelity of protein
synthesis.
2.3. Effects of modifications on the stability of h31
Absorbance versus temperature profiles (melting curves) were
obtained at pH 7.0 for all four RNA constructs. They were analyzed
in terms of D , D , DH°, D
Gꢀ37 Gꢀ50 S°, and melting temperature (Tm).33,34
The normalized absorbance plots at single RNA concentrations are
shown in Fig. 2. The melting curves are biphasic for four RNAs (see
Supplementary data) with transitions at 0–35 °C and 40–80 °C. The
low temperature transitions were concentration dependent, sug-
gesting the formation of a bimolecular complex, such as a duplex
or loop–loop interaction. The higher melting transitions were con-
centration independent, consistent with unimolecular unfolding of
a hairpin structure.35 The corresponding thermodynamic parame-
ters for the four RNAs are given in Table 1. The data indicate slight
destabilizing effects (DDG°37 values of 0.2–0.5 kcal/mol) of the
modifications on helix 31. The observed order of stability of the
2.4. Effects of modifications on the secondary structure of h31
The CD spectra of the helix 31 analogues were obtained to ana-
lyze the effects of modifications on the folded structure (Fig. 3). The
data indicate that the unmodified, singly modified, or doubly mod-
ified RNAs contain A-form stem regions, and they display similar
conformations. They all have peak maxima at 270 nm and minima
at 240 nm, similar to other A-form RNAs. A difference spectrum
was obtained using Eq. 1 in order to determine if the structural
changes induced by the modified nucleotides are additive. In Eq.

5890
N. D. Abeydeera, C. S. Chow / Bioorg. Med. Chem. 17 (2009) 5887–5893
As shown in Figure 3D, the total difference spectrum is close to
zero except for slight changes in the lower wavelength range
(<260 nm). Therefore, the data are interpreted as the presence of
modifications in h31 having subtle effects on the RNA structure.
Studies in K+- and Mg2+-containing buffers showed similar results
(Supplementary data).
3. Conclusions
Several conclusions can be drawn from these studies with re-
spect to the relative stabilities and structures of RNAs with single
and multiple modified nucleotides. Small reproducible differences
in the free energy values for the E. coli h31 variants reveal slight
destabilizing effects of the modifications on helix 31. A recent
X-ray crystal structure of the T. thermophilus 70S ribosome com-
plexed with a model mRNA and two tRNAs revealed that the posi-
tions of the 16S rRNA P-site nucleotides in the vacant ribosome
superimpose well with those in the tRNA-containing complex,
with the exception of m22G966.16 Residue m22G966 (m2G966 in
E. coli ribosomes) is flipped out in the crystal structure of the T.
thermophilus 70S ribosome containing a model mRNA and two
tRNAs (Fig. 4A)15,16 or remains stacked in the 30S ribosomal sub-
unit from T. thermophilus crystal structure (Fig. 4B).22 The interac-
tion with the anticodon loop of the P-site-bound tRNA appears to
be stabilized by stacking interactions involving m22G966 with ri-
bose 34. Hence, the flipped-out base has been suggested to facili-
tate correct positioning of the tRNA during translation.16,25
Therefore, the slight destabilizing effects of modifications in h31
may be important for facilitating the flipping movement of residue
966; but at the same time, stacking interactions with the tRNA are
stabilized through the methyl group. Positioned in the middle of
the three stacked bases, the m5C967 residue has a greater destabi-
lizing effect than m2G966 (ECh31M5C vs ECh31M2G). This result
may be due to a greater disruption of stacking by the methylated
base of m5C967.
The CD data indicate that the unmodified, singly modified, and
fully modified RNAs all contain A-form stem regions and display
similar conformations. Minor differences between the CD spectra
of the fully modified and unmodified h31 constructs indicate pos-
sible differences in the loop regions. These differences could arise
from modification-dependent changes in the loop, such as altered
base stacking at positions 966, 967, and 968. The UV melting data
reveal, however, that the presence of modifications at specific loca-
tions does not influence the ability of the constructs to form stable
hairpin loop structures.
The exact functional role of the modifications at positions 966
and 967 of h31 is still unknown. Ribosomes carry out the essential
biochemical process of translation, which requires an exquisite ar-
ray of highly specific interactions between rRNA, mRNA, tRNAs,
and ribosomal proteins. Modifications are believed to help fine-
tune ribosome function.3 Since proper ribosome function depends
on the correct balance of speed and accuracy of tRNA binding, pep-
tide-bond formation and tRNA release, methylations in h31 could
play a role in maintaining proper interactions within the ribo-
Figure 2. Representative normalized UV melting curves of the modified RNAs
compared to the unmodified RNA, taken in 15 mM NaCl, 20 mM sodium cacodylate,
0.5 mM Na2EDTA (pH 7.0), are shown. The melting curves for the unmodified RNA
(ECh31UNMOD, solid gray lines in panels A–C) are compared to those for the
modified RNAs (dashed lines): (A) ECh31WT, (B) ECh31M2G, and (C) ECh31M5C. All
of the melting curves were normalized at 95 °C and absorbance measurements
were taken at 280 nm.
1, the difference spectrum for the singly modified RNAs and
unmodified RNA is set equal to the difference spectrum of fully
modified (wild-type) and unmodified RNA. If the effects of the
modifications on the structure are additive, then the total differ-
ence spectrum should be equal to zero (Eq. 2).
ECh31M5C þ ECh31M2G ꢁ ð2 ꢂ ECh31UNMODÞ
¼ ECh31WT ꢁ ECh31UNMOD
difference ¼ ECh31M5C þ ECh31M2G
ꢁ ECh31UNMOD ꢁ ECh31WT
ð1Þ
ð2Þ
Table 1
Thermodynamics of the four RNA analogues
D
Gꢀ50 (kcal/mol)
D
Gꢀ37 (kcal/mol)
D
H° (kcal/mol)
D
S° (cal/K mol)
Tm (°C)
ECh31UNMOD
ECh31M5C
ECh31M2G
ECh31WT
ꢁ0.9a
ꢁ0.6a
ꢁ0.7a
ꢁ0.5a
ꢁ2.6 0.1
ꢁ2.2 0.1
ꢁ2.4 0.1
ꢁ2.1 0.1
ꢁ44.1 1.0
ꢁ40.5 0.9
ꢁ41.9 1.1
ꢁ37.5 2.2
ꢁ133.8 3.0
ꢁ123.3 3.1
ꢁ127.5 3.2
ꢁ114.4 5.8
57
55
56
56
Each measurement was taken in triplicate. Best fits were obtained by assuming a hairpin formation. The buffer conditions were 15 mM NaCl, 20 mM sodium cacodylate and
0.5 mM Na2EDTA, pH 7.0.
a
A conservative estimate of the standard error for
D
G°50 is 3% ( 0.2 kcal/mol).43

N. D. Abeydeera, C. S. Chow / Bioorg. Med. Chem. 17 (2009) 5887–5893
5891
4. Experimental
4.1. Synthesis of the 6-O-DPC-2-N-methylguanosine
4.1.1. General
Most reagents and solvents were either purchased from Aldrich
(St. Louis, MO) or Acros (Morris Plains, NJ) and used as received.
Anhydrous pyridine was purchased in a sure-seal bottle from
Aldrich. Methylene chloride (CH2Cl2) was distilled over CaH2.
Methanol and triethylamine were purchased dry from Aldrich in
a sure-seal bottle. Moisture sensitive reactions involved flame-dry-
ing equipment (syringes, round-bottom flasks, stir-bars, etc.) under
vacuum and performing reactions under dry argon. TLC analyses
were accomplished with precoated silica gel (0.25 mm thickness)
glass plates. Reactions were monitored by visualizing the TLC
plates under UV light and/or by staining with phosphomolybdic
acid (PMA) solution (10% w/v in absolute ethanol) and heating with
a hot plate. Flash chromatography was performed with Silica Gel
60 (0.038–0.063 mm). Flash columns were neutralized with 0.5–
1% triethylamine (TEA) prior to purification of pH sensitive inter-
mediates/compounds. 1H NMR and 13C NMR spectra were recorded
on either a Varian Unity 300, Mercury 400, or Varian Unity 500
spectrometer and referenced to tetramethylsilane as an internal
standard. ESI spectra were recorded on a Quattro LC (Bruker Dal-
tonics) in the positive ion mode unless otherwise noted.
4.1.2. 20,30,50-Tri-O-acetylguanosine (1)
Compound
1 was generated according to a literature
procedure.27
4.1.3. 20,30,50-Tri-O-acetyl-2-N-(p-
methylphenylthiomethyl)guanosine (2)
This compound was generated using a procedure described in
the literature.28
4.1.4. 20,30,50-Tri-O-acetyl-2-N-methylguanosine (3)
Compound 3 was prepared using a procedure described in the
literature.38
4.1.5. 2-N-Methyl-6-O-(diphenylcarbamoyl)guanosine (5)
To a solution of 20,30,50-tri-O-acetyl-2-N-methylguanosine 3
(3.0 g, 7.1 mmol, 1.0 equiv) in dry pyridine (50 mL) were added
diphenylcarbamoyl chloride (3.45 g, 15 mmol, 2.1 equiv) and diiso-
propylethylamine (1.9 mL, 11.4 mmol, 1.6 equiv). The dark brown
reaction mixture was then stirred at room temperature for 1 h to
Figure 3. CD spectra of the modified and unmodified RNA constructs are shown.
Each spectrum is an average of five scans. The CD spectrum of the unmodified
analogue (ECh31UNMOD, solid gray line) is shown in panels A–C with overlays
(dashed lines) of the ECh31M5C (A), ECh31M2G (B), and ECh31WT (C) RNAs. The
molar ellipticities are normalized to RNA concentrations. The total difference
spectrum (ECh31M5C + ECh31M2G ꢁ ECh31UNMOD ꢁ ECh31WT) is shown in
panel D (solid black line).
obtain
20,30,50-tri-O-acetyl-6-O-(diphenylcarbamoyl)-2-N-meth-
ylguanosine 4. TLC showed complete conversion to product at this
stage of the reaction. The reaction mixture was then diluted with
pyridine (15 mL) and EtOH (30 mL). To this solution cooled at
0 °C was added 2 M NaOH (25 mL), which was also precooled to
0 °C. The reaction mixture was stirred for 10 min at 0 °C. Acetic
acid (ca. 5 mL) was then added to neutralize the solution. Extrac-
tion with CH2Cl2 followed by chromatography on silica gel afforded
5 after two steps in 55% yield (1.92 g). TLC (CH2Cl2:MeOH, 9:1 v/v):
Rf = 0.5; ESI-MS (ES+) calcd for C24H24N6O6 492.1757, found 493.2
(M+H+), 515.2 (M+Na+), 1007.4 (2 M+Na+).
some.36,37 Mutational analyses revealed that a loss of methylation
at either position 966 or 967, leads to increased protein production
by the mutant ribosomes.21 Our data would therefore suggest that
methylations destabilize h31 in order to maintain the proper inter-
actions with tRNA, rRNA, or proteins. A lack of modification at res-
idues 966 or 967 in h31 could reduce the ability of base 966 to flip
and regulate tRNA affinity, positioning, or accuracy. Thus, it will be
of great interest to explore in greater detail the relationship be-
tween G966 and C967 methylation and translational fidelity. Fur-
thermore, the availability of a suitable method for synthesizing
m2G and its corresponding phosphoramidite (commercially not
available) will allow RNAs containing m2G modifications to be gen-
erated in sufficiently large quantities for use in additional biophys-
ical and ligand-binding studies.
4.2. Synthesis of 50-O-DMT-20-O-TOM-6-O-DPC-2-N-methylguano-
sine phosphoramidite
4.2.1. 50-O-(4,40-Dimethoxytrityl)-2-N-methyl-6-O-(diphenylcar-
bamoyl)guanosine (6)
Compound 5 (0.51 g, 1.05 mmol, 1.0 equiv) and 4,40-dimethoxy-
tritylchloride (0.39 g, 1.14 mmol, 1.09 equiv) were azeotroped
three times with toluene for ꢃ5 h. To the dried compound 5, anhy-

5892
N. D. Abeydeera, C. S. Chow / Bioorg. Med. Chem. 17 (2009) 5887–5893
Figure 4. Structures of h31 showing the flipped out (A) and stacked (B) conformations of residue G966 (PDB accession IDs—2J0015 and 1FJF22).
drous pyridine (5 mL) was added. The mixture was stirred at room
temperature under Ar atmosphere for 4 h. 4-Dimethylaminopyri-
dine (0.09 g, 0.7 mmol, 0.7 equiv) was subsequently added and
stirring was continued for 17 h. The reaction was quenched with
methanol (1 mL) and evaporated to dryness. The crude residue
was dissolved in 50 mL of dichloromethane and washed with 5%
sodium bicarbonate followed by saturated sodium chloride. The
organic layer was dried over sodium sulfate and filtered. The prod-
uct was then purified by silica gel chromatography using a solvent
mixture of 90% dichloromethane, 9% methanol, 1% triethylamine to
give 6 as a light yellow crystalline solid (0.56 g, 67%). TLC
(CH2Cl2:MeOH, 9:1 v/v): Rf = 0.4; ESI-MS (ES+) calcd for
C45H42N6O8 794.3, found 795.7 (M+H+), 303.4 ([(MeO)2Tr]+).
dichloromethane. Next, N,N-diisopropylethylamine (0.3 mL,
2.5 mmol, 10 equiv) and 2-cyanoethyldiisopropylchlorophosph-
oramidite (0.08 mL, 0.38 mmol, 1.5 equiv) were added and the
mixture was stirred for 2 h at room temperature. The reaction
was quenched with 5% aqueous sodium bicarbonate, and then it
was extracted with 2 ꢂ 50 mL of dichloromethane. Combined ex-
tracts were dried over anhydrous sodium sulfate and evaporated.
The crude mixture was purified by silica gel column chromatogra-
phy (hexane:ethyl acetate, 3:1 and triethylamine, 0.5%) to yield 8
as
a white form (0.285 g, 95%). TLC (hexane:EtOAc:Et3N =
75%:24.5%:0.5%): Rf = 0.23; 1H NMR ((CD3)2CO, 400 MHz) (mixture
of diastereoisomers) 0.89–1.05 (2 m, 60H), 1.18–1.30 (2 m, 10H),
1.45 (t, 4H), 2.49 (br s, 2H), 2.79–2.84 (m, 8H), 3.39–3.41 (m,
2H), 3.66–3.75 (m, 14H), 4.17–4.19 (m, 2H), 4.69 (m, 2H), 5.12–
5.14 (m, 8H), 6.11 (d, J = 5.2 Hz, 2H), 6.79–6.83 (m, 10H), 7.17–
7.50 (2 m, 36H), 8.04 (s, 2H); 13C NMR ((CD3)2CO, 400 MHz)
12.56, 18.04, 18.08, 20.7, 20.75, 24.76, 24.82, 24.89, 24.95, 28.97,
43.82, 43.91, 43.96, 44.06, 55.45, 59.0, 59.16, 59.89, 60.02, 64.39,
72.59, 84.4, 84.7, 87.14, 87.71, 90.03, 90.36, 113.86, 127.54,
128.54, 128.58, 128.91, 128.98, 129.91, 130.85, 130.93, 130.99,
136.57, 136.69, 143.38, 145.96, 151.43, 157.33, 159.58, 160.82,
206.15; 31P NMR ((CD3)2CO, 400 MHz) (mixture of diastereoiso-
mers) 150.96, 151.14; High resolution ESI-MS (ES+) calcd for
C64H81N8O10PSi 1180.5583, found 1181.5634 (M+H+), 1203.5439
(M+Na+), 1219.5358 (M+K+).
4.2.2. 50-O-(4,40-Dimethoxytrityl)-20-O-[[(triisopropylsilyl)oxy]-
methyl]-2-N-methyl-6-O-(diphenylcarbamoyl) guanosine (7)
Di-tert-butyltindichloride (0.26 g, 0.846 mmol, 1.2 equiv) was
added to a solution of dry 1,2-dichloroethane (6.5 mL) containing
compound 6 (0.56 g, 0.705 mmol, 1.0 equiv) and ethyldiisopropyl-
amine (0.36 mL, 2.82 mmol, 4.0 equiv). The reaction mixture was
heated to 70 °C for 15 min under refluxing conditions. Then, the
mixture was allowed to cool to room temperature. Upon cooling
down, the mixture became cloudy and light brown in color. The
crude reaction mixture was then stirred with [(triisopropylsi-
lyl)oxy]methylchloride (0.18 mL, 0.776 mmol, 1.1 equiv) for 3 h
at room temperature. After 3 h, the mixture was evaporated to dry-
ness. The resulting crude residue was dissolved in 20 mL of dichlo-
romethane and washed with saturated sodium bicarbonate
followed by saturated sodium chloride. The organic layer was dried
over sodium sulfate and filtered. The product was then purified by
silica gel column chromatography using a solvent mixture of
dichloromethane:methanol (20:1) and triethylamine (0.5%) to give
7 as a light yellow oil (0.49 g, 70%). TLC (CH2Cl2:MeOH, 9:1 v/v):
Rf = 0.7; ESI-MS (ES+) calcd for C55H64N6O9Si 980.4, found 981.9
(M+H+), 1003.9 (M+Na+), 1019.9 (M+K+).
4.3. RNA oligonucleotide synthesis
The four RNA hairpins used in this study (Fig. 1) were chemi-
cally synthesized at the W. M. Keck Foundation at Yale University,
New Haven, Connecticut, USA. For RNAs containing m2G, 50
lmol
of the corresponding phosphoramidite were provided. The m5C
phosphoramidite was purchased from Glen Research. The se-
quences of the four synthetic RNAs are as follows (U, C, G, A are
nucleotides representing the natural E. coli sequence; g, c are added
nucleotides to enhance hairpin stability):
4.2.3. 50-O-(4,40-Dimethoxytrityl)-20-O-[[(triisopropylsilyl)oxy]-
methyl]-2-N-methyl-6-O-(diphenylcarbamoyl) guanosine 30-(2-
cyanoethyl diisopropylphosphoramidite) (8)
Compound 7 (0.25 g, 0.25 mmol, 1.0 equiv) was dried exten-
sively under vacuum. Then, it was dissolved in 5 mL of anhydrous
50-gUU CGA UGC AAC GCG AAc-30 (ECh31UNMOD)
50-gUU CGA UG(m5C) AAC GCG AAc-30 (ECh31M5C)
50-gUU CGA U(m2G)C AAC GCG AAc-30 (ECh31M2G)
50-gUU CGA U(m2G)(m5C) AAC GCG AAc-30 (ECh31WT)

N. D. Abeydeera, C. S. Chow / Bioorg. Med. Chem. 17 (2009) 5887–5893
5893
4.4. RNA deprotection and purification
References and notes
1. Brimacombe, R.; Mitchell, P.; Osswald, M.; Stade, K.; Bochkariov, D. FASEB J.
1993, 7, 161.
2. Agris, P. F. Prog. Nucleic Acid Res. 1996, 53, 79.
Upon completion of coupling on an automated synthesizer, the
CPG-boundRNA was cleavedfrom the solid support and deprotected
with 1:3 (v/v) EtOH/NH4OH and TBAF (tetrabutylammonium fluo-
3. Decatur, W. A.; Fournier, M. J. Trends Biochem. Sci. 2002, 27, 344.
4. Chow, C. S.; Lamichhane, T. N.; Mahto, S. K. ACS Chem. Biol. 2007, 2, 610.
5. Cunningham, P. R.; Richard, R. B.; Weitzmann, C. J.; Nurse, K.; Ofengand, J.
Biochimie 1991, 73, 789.
6. Denman, R.; Weitzmann, C.; Cunningham, P. R.; Negre, D.; Nurse, K.; Colgan, J.;
Pan, Y. C.; Miedel, M.; Ofengand, J. Biochemistry 1989, 28, 1002.
7. Denman, R.; Negre, D.; Cunningham, P. R.; Nurse, K.; Colgan, J.; Weitzmann, C.;
Ofengand, J. Biochemistry 1989, 28, 1012.
29,39
ride solution, 1 M in THF) as described in the literature.
The
RNAs were desalted over Poly-Pak II cartridges (Glen Research), then
purified by HPLC on an XTerra MS C18 column (2.5
m, 10 ꢂ 50 mm,
l
Waters) in which the eluent was 0.1 M TEAA (triethylammonium
acetate) buffer, pH 7.0, with a 5–15% linear gradient of acetonitrile
over 25 min at a flow rate of 4.0 mL/min. After HPLC purification,
each oligomer was further desalted by ethanol precipitation and
dialysis for 3 days against RNase-free, deionized water using a
1000 molecular weight cut-off membrane (Spectra-Por). RNA con-
centrations were calculated using Beer’s law and a single-stranded
8. Green, R.; Noller, H. F. RNA 1996, 2, 1011.
9. Starr, J. L.; Fefferman, R. J. Biol. Chem. 1964, 239, 3457.
10. Ehresmann, C.; Stiegler, P.; Mackie, G. A.; Zimmermann, R. A.; Ebel, J. P.; Fellner,
P. Nucleic Acids Res. 1975, 2, 265.
11. Ehresmann, C.; Stiegler, P.; Fellner, P.; Ebel, J. P. Biochimie 1975, 57, 711.
12. Yusupov, M. M.; Yusupova, G. Z.; Baucom, A.; Lieberman, K.; Earnest, T. N.;
Cate, J. H.; Noller, H. F. Science 2001, 292, 883.
13. Aduri, R.; Psciuk, B. T.; Saro, P.; Taniga, H.; Schlegel, H. B.; SantaLucia, J. J. Chem.
Theory Comput. 2007, 3, 1464.
14. Doring, T.; Mitchell, P.; Osswald, M.; Bochkariov, D.; Brimacombe, R. EMBO J.
1994, 13, 2677.
extinctioncoefficient(e . Thesameextinction
)of176,900 Mꢁ1 cmꢁ1 40
coefficients were used for guanosine and m2G (1.4 ꢂ 104 Mꢁ1 cmꢁ1
at pH 7.0) and cytidine and m5C (9.1 ꢂ 103 Mꢁ1 cmꢁ1 at pH 7.0).
15. Selmer, M.; Dunham, C. M.; Murphy, F. V., IV; Weixlbaumer, A.; Petry, S.;
Kelley, A. C.; Weir, J. R.; Ramakrishnan, V. Science 2006, 313, 1935.
16. Korostelev, A.; Trakhanov, S.; Laurberg, M.; Noller, H. F. Cell 2006, 126, 1065.
17. Berk, V.; Zhang, W.; Pai, R. D.; Cate, J. H. D. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
15830.
18. Cannone, J. J.; Subramanian, S.; Schnare, M. N.; Collett, J. R.; D’Souza, L. M.; Du,
Y.; Feng, B.; Lin, N.; Madabusi, L. V.; Müller, K. M.; Pande, N.; Shang, Z.; Yu, N.;
Gutell, R. R. BMC Bioinformatics 2002, 3, 2.
19. Jemiolo, D. K.; Taurence, J. S.; Giese, S. Nucleic Acids Res. 1991, 19, 4259.
20. Lesnyak, D. V.; Osipiuk, J.; Skarina, T.; Sergiev, P. V.; Bogdanov, A. A.; Edwards,
A.; Savchenko, A.; Joachimiak, A.; Dontsova, O. A. J. Biol. Chem. 2007, 282, 5880.
21. Saraiya, A. A.; Lamichhane, T. N.; Chow, C. S.; SantaLucia, J., Jr.; Cunningham, P.
R. J. Mol. Biol. 2008, 376, 645.
4.5. UV melting studies
The absorbance versus temperature profiles were obtained on
an Aviv 14DS UV–vis spectrophotometer with a five-cuvette ther-
moelectric controller. Microcuvettes with two different path-
lengths, 0.1 and 0.2 cm (60 and 120 lL volumes, respectively),
were employed. Each measurement was taken in triplicate. The
buffer used in each experiment contained 15 mM NaCl, 20 mM so-
dium cacodylate, and 0.5 mM Na2EDTA (pH 7.0), unless noted
otherwise. Each oligomer was dissolved in a specific volume to
yield an absorbance reading just below 2.0 in a 0.1 cm pathlength
cuvette. The RNA concentrations were determined from the absor-
bance values (260 nm) at 95 °C. The absorbance data were col-
lected at 280 nm from 0 to 95 °C with a constant heating rate of
0.5 °C/min.41 Thermodynamic parameters were obtained from
the absorbance versus temperature profiles using the MELTWIN v.
3.5 melting curve program.33 This program performs a van’t Hoff
analysis, assuming a two-state model for the transition between
a native and a denatured (random coil) structure of a hairpin loop.
22. Brodersen, D. E.; Clemons, W. M., Jr.; Carter, A. P.; Wimberly, B. T.;
Ramakrishnan, V. J. Mol. Biol. 2002, 316, 725.
23. Schuwirth, B. S.; Borovinskaya, M. A.; Hau, C. W.; Zhang, W.; Vila-Sanjurjo, A.;
Holton, J. M.; Cate, J. H. D. Science 2005, 310, 827.
24. Moazed, D.; Noller, H. F. J. Mol. Biol. 1990, 211, 135.
25. Selmer, M.; Dunham, C. M.; Murphy, F. V. t.; Weixlbaumer, A.; Petry, S.; Kelley,
A. C.; Weir, J. R.; Ramakrishnan, V. Science 2006, 313, 1935.
26. Baxter-Roshek, J. L.; Petrov, A. N.; Dinman, J. D. PLoS ONE 2007, 2, e174.
27. Matsuda, A.; Shinozaki, M.; Suzuki, M.; Watanabe, K.; Miyasaka, T. Synthesis-
Stuttgart 1986, 385.
28. Bridson, P. K.; Reese, C. B. Bioorg. Chem. 1979, 8, 339.
29. Kamimura, T.; Tsuchiya, M.; Urakami, K.; Koura, K.; Sekine, M.; Shinozaki, K.;
Miura, K.; Hata, T. J. Am. Chem. Soc. 1984, 106, 4552.
30. Koizumi, M.; Akahori, K.; Ohmine, T.; Tsutsumi, S.; Sone, J.; Kosaka, T.; Kaneko,
M.; Kimura, S.; Shimada, K. Bioorg. Med. Chem. Lett. 2000, 10, 2213.
31. Höbartner, C.; Kreutz, C.; Flecker, E.; Ottenschläger, E.; Pils, W.; Grubmayr, K.;
Micura, R. Monatsh. Chem. 2003, 134, 851.
32. Boudou, V.; Langridge, J.; Van Aerschot, A.; Hendrix, C.; Millar, A.; Weiss, P.;
Herdewijn, P. Helv. Chim. Acta 2000, 83, 152.
33. McDowell, J. A.; Turner, D. H. Biochemistry 1996, 35, 14077.
34. SantaLucia, J., Jr.; Kierzek, R.; Turner, D. H. Science 1992, 256, 217.
35. Tinoco, I., Jr.; Sauer, K.; Wang, J. C. Physical Chemistry, Principles and Applications
in Biological Sciences. Prentice-Hall: Englewood Cliffs, NJ, 1985.
36. Gromadski, K. B.; Daviter, T.; Rodnina, M. V. Mol. Cell 2006, 21, 369.
37. Fahlman, R. P.; Dale, T.; Uhlenbeck, O. C. Mol. Cell 2004, 16, 799.
38. Kemal, O.; Reese, C. B. Synthesis 1980, 12, 1025.
4.6. Circular dichroism (CD) spectroscopy
CD spectra were obtained on an Applied Photophysics Chirascan
circular dichroism spectrometer (220–320 nm) at 25 °C in 15 mM
NaCl, 20 mM sodium cacodylate, and 0.5 mM Na2EDTA at pH 7.0.
The RNA concentrations were maintained at 2.5–3.0
CD experiments. Based on the RNA strand concentration, the mea-
sured CD spectra were converted to molar ellipticity (
),42 which
lM for all
D
e
denotes the moles of RNA molecules rather than moles of individ-
ual residues present in the sequence.
39. Pitsch, S.; Weiss, P. A.; Jenny, L.; Stutz, A.; Wu, X. Helv. Chim. Acta 2001, 84,
3773.
Acknowledgments
40. Richards, E. G. In Handbook of Biochemistry and Molecular Biology; CRC Press:
Cleveland, OH, 1975; p 596.
41. SantaLucia, J., Jr. In Spectrometry and Spectrofluorometry; Gore, M. G., Ed.;
Oxford University Press: Oxford, UK, 2000; p. 329.
42. Cantor, C. R., Schimmel, P. R. In Biophysical Chemistry. Part II; W. H. Freeman
and Co.: San Francisco, CA, 1980; p 412.
We thank Tek Lamichhane, John SantaLucia, and Philip Cunn-
ingham for helpful discussions and Lew Hryhorczuk, Andrew Feig,
and Norman Watkins for technical assistance. This work was sup-
ported by the National Institutes of Health (AI061192).
43. SantaLucia, J., Jr.; Turner, D. H. Biopolymers 1997, 44, 309.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.07.008.