Probing the stabilizing effects of modified nucleotides in the bacterial decoding region of 16S ribosomal RNA

Article abstract of DOI:10.1016/j.bmc.2013.03.010

Abstract The bacterial decoding region of 16S ribosomal RNA has multiple modified nucleotides. In order to study the role of N⁴,2′-O- dimethylcytidine (m⁴Cm), the corresponding phosphoramidite was synthesized utilizing 5′-silyl-2′-AC

Full text of DOI:10.1016/j.bmc.2013.03.010

Bioorganic & Medicinal Chemistry 21 (2013) 2720–2726
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Probing the stabilizing effects of modified nucleotides
in the bacterial decoding region of 16S ribosomal RNA
⇑
Santosh K. Mahto, 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
Article history:
The bacterial decoding region of 16S ribosomal RNA has multiple modified nucleotides. In order to study the
role of N4,20-O-dimethylcytidine (m4Cm), the corresponding phosphoramidite was synthesized utilizing 50-
silyl-20-ACE chemistry. Using solid-phase synthesis, m4Cm, 5-methylcytidine (m5C), 3-methyluridine
(m3U), and 20-O-methylcytidine (Cm) were site-specifically incorporated into small RNAs representing
the decoding regions of different bacterial species. Biophysical studies were then used to provide insight
into the stabilizing roles of the modified nucleotides. These studies reveal that methylation of cytidine
and uridine has different effects. The same modifications at different positions or sequence contexts within
similar RNA constructs also have contrasting roles, such as stabilizing or destabilizing the RNA helix.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 31 December 2012
Revised 3 March 2013
Accepted 7 March 2013
Available online 21 March 2013
Keywords:
Ribosomal RNA modification
Decoding region
N4,20-O-Dimethylcytidine
5-Methylcytidine
3-Methyluridine
1. Introduction
m4Cm and m3U are commonly found at positions 1402 and 1498,
respectively, in the decoding region of various other bacterial spe-
The ribosome carries out protein synthesis, an essential process
for life. The key steps of protein translation include decoding of the
mRNA three-nucleotide codons with the correct tRNA anticodons,
followed by peptide bond formation.1 The decoding region (helix
44) is a very important functional region of the 16S ribosomal
RNA (rRNA), which is involved in translational accuracy (decoding)
to synthesize proteins in an error-free manner.1 This region is in-
volved in the selection of initiator tRNA, as well as cognate amino-
acyl-tRNAs, according to the mRNA sequence. The decoding region
is involved in binding to mRNA, tRNAs, and the large subunit
rRNA.2–5 This region is also responsible for translocation.6 In addi-
tion, several known antibiotics such as aminoglycosides bind to the
decoding region.7
Modified nucleotides are a universal feature of ribosomal RNAs.
Over one hundred modified nucleotides have been discovered in
RNA,8 which enhance the diverse structural and functional ability
of RNA.9 A wide variety of modified nucleotides are present at or
near the functional sites of the ribosome.10 The modified
nucleotides N4,20-O-dimethylcytidine (m4Cm), 5-methylcytidine
(m5C), and 3-methyluridine (m3U) are found in the bacterial
decoding region of 16S rRNA. The modified nucleotide m4Cm is a
unique nucleotide only found to date in helix 44.11–16 The modified
nucleotides m4Cm at position 1402, m5C at 1407, and m3U at 1498
are found in Escherichia coli 16S rRNA. The modified nucleotides
cies; whereas, the nucleotide m5C varies in number and position in
different bacterial decoding regions. In Clostridium acetobutylicum,
m5C is found at 1409; in Thermus thermophilus, there are three m5C
residues at 1400, 1404 and 1407; and in Thermotoga maritime,
there is no m5C in the decoding region, but a Cm residue occurs
at 1409 (E. coli numbering).14–16
The nucleoside m4Cm was isolated from E. coli and character-
ized by Nichols and Lane in 1966.17,18 The modified nucleosides
m5C and m3U were first isolated by Amos and Korn in 1958 and
Hall in 1963.19,20 RsmF and RsmE are specific methyltransferase
enzymes that post-transcriptionally methylate pyrimidines to gen-
erate m5C1407 and m3U1498, respectively.21,22 The methyltrans-
ferase enzymes RsmH and RsmI are responsible for N4-base and
20-O-sugar methylation of m4Cm1402.23
The fact that modified nucleotides are present in the biologi-
cally significant decoding region implies that they have important
roles; however, these roles are still not very well understood. A
mutational analysis of conserved bases m4Cm1402 and A1500 in
16S rRNA (E. coli) revealed that these nucleotides may be involved
in important tertiary interactions.24 Crystal structures of the ribo-
some show m4Cm and m3U hydrogen bonded with mRNA.4,5 Fur-
thermore, there is a metal ion that bridges m4Cm and G1401 to
the codon.5 Another report suggested a connection between the
ribosomal modifications and initiator tRNA selection.25 Previous
studies provided insight into the effects of functional group modi-
fications on nucleoside structure.26 The next step is to determine
the role of modified nucleotides within the context of small rRNA
⇑
Corresponding author. Tel.: +1 313 577 2594; fax: +1 313 577 8822.
E-mail addresses: csc@chem.wayne.edu, cchow@wayne.edu (C.S. Chow).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.010

S. K. Mahto, C. S. Chow / Bioorg. Med. Chem. 21 (2013) 2720–2726
2721
Scheme 1. Synthesis of 50-O-benzhydryloxy-bis(trimethylsilyloxy)silyl-30-O-(N,N-
diisopropylamino)methoxyphosphonyl-N4,20-O-dimethylcytidine: Reagents and
conditions: (i) benzhydryloxy-bis(trimethysilyloxy)chlorosilane (BzH-Cl), DMF–
CH2Cl2, 0 °C, 3 h; (ii) bis(N,N-diisopropylamino)-methoxyphosphine, 1H-tetrazole,
CH2Cl2, rt, overnight.
The 50-BzH protective group was introduced to m4Cm with
benzhydryloxy-bis(trimethylsilyloxy)chlorosilane (BzH-Cl) and
N,N-diisopropylamine at 0 °C for 3 h to afford compound 1 in 80%
yield. The phosphoramidite was obtained by the addition of
bis(N,N-diisopropylamino)methoxyphosphine and 1H-tetrazole to
a CH2Cl2 solution of compound 1 at room temperature. The final
product 50-O-benzhydryloxy-bis(trimethylsilyloxy)silyl-30-O-(N,N-
diisopropylamino)methoxyphosphonyl-N4,20-O-dimethylcytidine (2)
was obtained in 75% yield from 1. The m4Cm phosphoramidite
was synthesized in overall 60% yield in two steps from m4Cm.
The Cm phosphoramidite is commercially available.
Figure 1. The decoding region model system is shown with indication of the
modified nucleotide identity and positions. Nucleotide numbers are shown in
parenthesis (E. coli numbering). The native decoding sequence is boxed, whereas
additional nucleotides outside the box are used to stabilize the duplex structure
(clamp).
2.2. Solid-phase synthesis of decoding region RNA
The m4Cm, m5C, m3U, and Cm modified nucleotide phospho-
ramidites along with standard A, C, G and U phosphoramidites were
used to synthesize RNAs containing site-specific modifications by
models, which will help to further understand their roles within
the complete ribosome.
In order to understand the roles of modified nucleotides within
the context of a functionally important model RNA system repre-
senting the decoding region of 16S rRNA, they were site-specifi-
cally incorporated into the target by using chemical synthesis. A
modified nucleotide phosphoramidite of m4Cm was generated
and used in solid-phase synthesis, along with Cm, m5C, m3U, and
standard nucleotide phosphoramidites, to produce a series of bac-
terial decoding region model systems (Fig. 1). These analogues
were constructed by a mix-and-match approach, using a set of
50- and 30-half single-stranded oligonucleotides. The stabilizing
roles of the modified nucleotides in the decoding region RNAs were
then determined by using UV melting. A related decoding region
model system without modifications was used previously for li-
gand interactions and shown to be a good representation of the na-
tive rRNA within the context of complete ribosomes.27–30 The work
in this study reveals that the natural pyrimidine modifications
have a subtle effect on stability of this functionally important re-
gion of the ribosome.
50-BzH-20-ACE solid-phase synthesis on the
lmole scale. Nine dif-
ferent RNA strands were synthesized (Supplementary data,
Table S1) by utilizing the 50-silyl-20-ACE protocol and deprotected
according to literature procedures.33–35 Eleven different decoding
region model systems were generated from the single strands
(Fig. 2), such that the individual effects of the modified nucleotides
2. Results and discussion
2.1. Phosphoramidite synthesis
Chemical synthesis was employed for the site-specific incorpo-
ration of modified nucleotides into the small (21–24 nucleotide)
rRNA constructs. Among several available approaches, the phos-
phoramidite method is the most frequently used.31 Synthesis of
the modified nucleotide m4Cm phosphoramidite was accom-
plished using 50-BzH-20-ACE protection strategies (Scaringe meth-
od).32 This method has several distinct advantages, such as mild
deprotection, higher coupling efficiencies, and shorter coupling
times for small oligonucleotide constructs. The modified nucleo-
side m4Cm has methyl groups at the N4 and 20-O positions; hence,
exocyclic amine and 20-ACE-orthoester protection were not re-
quired. Therefore, only two steps were required for synthesis of
the m4Cm phosphoramidite (Scheme 1) instead of five steps as
needed for the standard 50-BzH-20-ACE nucleotides.
Figure 2. The decoding region model systems representing unmodified C-C-U,
m4Cm-C-U, C-m5C-U, C-C-m3U, Cm-C-U, m4Cm-m5C-U, m4Cm-C-m3U, C-m5C-m3U,
Clostridium acetobutylicum, and Thermus thermophilus are shown with E. coli
numbering. The modified nucleotides m4Cm and m3U are found at positions 1402
and 1498. The modified nucleotide m5C is found at positions 1407 and 1409 in
E. coli and C. acetobutylicum, respectively. There are three m5C residues at positions
1400, 1404, and 1407 in T. thermophilus.

2722
S. K. Mahto, C. S. Chow / Bioorg. Med. Chem. 21 (2013) 2720–2726
could be studied. Nine different decoding region E. coli rRNA con-
structs were generated, namely unmodified (C-C-U), singly modi-
fied (Cm-C-U, m4Cm-C-U, C-m5C-U, and C-C-m3U), doubly
modified (m4Cm-m5C-U, m4Cm-C-m3U, and C-m5C-m3U), and tri-
ply modified (m4Cm-m5C-m3U, wild type, Fig. 1), in which the three
symbols indicate the nucleotide composition at positions 1402,
1407 and 1498, respectively. The Cm-C-U decoding region RNA
was generated in order to determine the effects of sugar-only mod-
ification. The C. acetobutylicum and T. thermophilus decoding region
RNAs were synthesized so that the effects of modified nucleotides
at different positions or additional locations could be studied.
intermediate stability, suggesting that the stabilizing effects are
additive.
The modified nucleotide m3U has a negligible effect on the
decoding region stability in the case of the singly modified con-
struct, C-C-m3U. This result is confirmed by comparing the
D
Gꢀ37
values for C-m5C-U and C-m5C-m3U or m4Cm-m5C-U and m4Cm-
m5C-m3U, in which m3U has little impact (Table 1). In contrast,
the combinations of m3U and/or m5C with m4Cm (m4Cm-C-m3U,
m4Cm-m5C-U or m4Cm-m5C-m3U) cause the stabilizing effects of
m4Cm to be diminished. In this case, the effects do not appear to
be additive, and instead indicate cooperativity of the modifications.
These differences are likely the result of unique structural effects of
cytidine and uridine methylation. Overall, the three modified
nucleotides have distinct, yet at the same time subtle, effects on
the decoding region stability.
2.3. UV-melting studies
Thermal melting studies were carried out on purified RNAs in
cacodylate buffer (20 mM sodium cacodylate, 1 M NaCl, and
0.5 mM EDTA buffer, pH 7.0). UV-absorbance values of the 50-
and the 30-half single-stranded decoding region RNA samples were
determined at 90 °C, and the strands were mixed in equimolar ra-
tios to obtain the duplex decoding regions. Absorbance versus tem-
perature profiles (UV-melting curves) were obtained for all of the
RNA constructs at five different concentrations per duplex. The
Meltwin 3.5 program was used to derive the thermodynamic
In order to examine more closely the stabilizing effect of m4Cm,
the sugar methylated analogue Cm was analyzed. The
D
Gꢀ37 value
for Cm-C-U is compared with those for C-C-U and m4Cm-C-U RNAs
(Table 1). The DDGꢀ37 value reveals that Cm is stabilizing relative to
cytidine, but less stabilizing than dimethylated cytidine, m4Cm.
This result shows that the stabilizing effect of m4Cm comes from
both sugar and base methylation. The m4C phosphoramidite was
not available; therefore, the stabilizing effect of base methylation
alone was not examined directly.
parameters (D , D D
Gꢀ37 Hs, Ss, and Tm).36 Each melting experiment
Our initial studies were carried out on the E. coli decoding region
analogues. It was of interest to consider the effects of m5C at differ-
ent positions of the decoding region, since this modification is pres-
ent at different positions, as well as in varying numbers in other
organisms. In C. acetobutylicum, m5C is present at position 1409 in-
stead of 1407 as in E. coli. When comparing the stabilities of the two
decoding regions, it has been found that there is no noticeable dif-
ference (Table 1). In contrast, there is a slight stabilizing effect in the
T. thermophilus construct compared to the E. coli decoding region;
the former has three m5C nucleotides in its decoding region com-
pared to only one in the latter (Table 1). The two additional m5C
nucleotides occur at positions 1400 and 1404 of T. thermophilus
(Fig. 2). The modified nucleotide m5C present at position 1407
was shown to have a small destabilizing effect in the E. coli con-
struct. Thus, the additional m5C nucleotides at positions 1400 and
1404 have stabilizing effects to provide overall greater stability to
the T. thermophilus analogue compared to the E. coli decoding re-
gion. Therefore, as was observed previously with pseudouridine,38
the effects of nucleotide (m5C) modification depend on the loca-
tions of those modifications within the RNA construct.
was done in triplicate, and the derived thermodynamic values
were averaged. The modified and unmodified decoding region ana-
logues were also studied in KCl buffer (20 mM cacodylic acid, 1 M
KCl, and 0.5 mM EDTA, pH 7.0), but no differences were observed.
The melting data for the unmodified decoding region were com-
pared to data from all other RNA constructs. The difference of
change in free energy (DDGꢀ37) was calculated by Eq. 1, in which
(A) is the modified construct and (B) is the unmodified decoding
region (C-C-U) construct:
DDGꢀ37
¼
D
Gꢀ37ðAÞ ꢁ
D
Gꢀ37ðBÞ
ð1Þ
Analysis of the UV-melting data of various decoding region con-
structs reveals information about the stabilizing roles of the mod-
ified nucleotides (Table 1). The duplexes are all relatively stable,
with
D
Gꢀ37 values ranging from ꢁ10.5 to ꢁ12.2 kcal/mol. Note that
this 16% difference in
D
Gs values is significant compared to the
smaller error of the experiment (2%).37 Comparison of the UV-
melting data (Table 1) for the m4Cm-C-U decoding region with
the unmodified decoding region C-C-U gives a DDGꢀ37 value of
ꢁ1.3 kcal/mol, revealing that m4Cm has a stabilizing role com-
pared to C. In contrast, m5C has a slightly destabilizing role; when
the decoding region C-m5C-U is compared with the unmodified
decoding region C-C-U, the DDGꢀ37 value is 0.6 kcal/mol. The
m4Cm nucleotide is stabilizing, m5C is slightly destabilizing, and
the resultant doubly modified decoding region m4Cm-m5C-U has
2.4. Circular dichroism studies
In order to examine the effects of modified nucleotides on the
overall conformation of the bacterial decoding region, circular
dichroism (CD) studies of the various decoding region analogues
Table 1
UV-melting data for decoding region analogues
Gꢀ37 (kcal/mol)
(
DDGꢀ37) (kcal/mol)
Analogues
D
Hs (kcal/mol)
D
Ss (cal/K mol)
Tm (°C)
(
D
E. coli analogues
C-C-U
ꢁ10.9 0.2
ꢁ12.2 0.2
ꢁ10.3 0.2
ꢁ10.7 0.2
ꢁ11.5 0.2
ꢁ11.4 0.2
ꢁ11.3 0.2
ꢁ10.5 0.2
ꢁ11.3 0.2
ꢁ11.4 0.2
ꢁ11.7 0.2
0
ꢁ49
ꢁ61
ꢁ48
ꢁ51
ꢁ55
ꢁ58
ꢁ55
ꢁ47
ꢁ57
ꢁ58
ꢁ61
5
4
5
7
5
7
7
6
7
7
6
ꢁ122 15
ꢁ156 13
ꢁ120 17
ꢁ129 21
ꢁ141 17
ꢁ149 21
ꢁ141 21
ꢁ119 20
ꢁ148 21
ꢁ149 21
ꢁ159 20
65.6
68.9
63.6
63.3
66.3
66.1
66.3
65.4
65.6
66.1
66.3
m4Cm-C-U
ꢁ1.3
C-m5C-U
0.6
C-C-m3U
0.2
Cm-C-U
ꢁ0.6
ꢁ0.5
ꢁ0.4
0.4
ꢁ0.4
ꢁ0.5
ꢁ0.8
m4Cm-m5C-U
m4Cm-C-m3U
C-m5C-m3U
m4Cm-m5C-m3U
C. acetobutylicum
T. thermophilus
Buffer conditions: 20 mM sodium cacodylate, 1 M NaCl, 0.5 mM Na2EDTA, pH 7.0.

S. K. Mahto, C. S. Chow / Bioorg. Med. Chem. 21 (2013) 2720–2726
2723
2.5. The roles of methylation in bacterial decoding region RNAs
In a crystal structure of E. coli ribosomes,40 m4Cm is stacked be-
tween G and C nucleotides. The thermal melting data reveal that
m4Cm has a stabilizing role. Hence, methylation likely increases
base-stacking interactions with the neighboring nucleotides. In
contrast, the modified nucleotide m5C at position 1407 is part of
a specific A-site motif in which two key residues, A1492 and
A1493, are highly dynamic.41 The modified nucleotide m5C has a
slight destabilizing effect, suggesting that its role might involve
enhancing the dynamic nature of the A-site motif. The modified
nucleotide m3U has only subtle effects on the decoding region sta-
bility. Therefore, m3U may have other roles in regulation of ribo-
some function. The combined modifications appear to have
additive effects, such that the overall impact on structure and sta-
bility is quite small. Nonetheless, these modifications are known to
influence the RNA structure and function within specific locations
of the decoding region.
The modified nucleotide m5C is present at different locations
within the bacterial decoding regions and also varies in number.
Compared to the E. coli decoding region, the T. thermophilus RNA
has two additional m5C nucleotides at positions 1400 and 1404,
which have slight stabilizing effects. Thus, our results show that
the same modified nucleotide at different locations of the decoding
region has opposing effects on stability, which was also observed
previously with the modified nucleotide pseudouridine.38 These
modified nucleotides are located at a functionally significant region
of the ribosome, suggesting that they may play a role in fine-tuning
the RNA structure and function.10 The fact that m4Cm and m3U are
hydrogen bonded with mRNA in ribosome crystal structures,4,5 and
m4Cm1402 and A1500 are at the center of the decoding domain
involving tertiary interactions with helix 45,24 suggests that the
modified nucleotides have roles in binding mRNA and assisting in
maintenance of tertiary interactions for proper ribosomal function.
Regarding studies with the decoding region analogues from other
species, it should be noted that T. thermophilus lives at high temper-
atures (80 °C), and rRNAs from such thermophiles contain a high
number of modifications.14 The m5C nucleotide has an effect on
decoding region stability, and therefore may play a role in mainte-
nance of translational fidelity in these organisms.
The knockout of methyltransferase genes (rsmE, rsmF, rsmH and
rsmI) that are responsible for modifications in the decoding region
leads to growth defects in bacteria. The doubling time of the meth-
yltransferase RsmE knockout strain, which is lacking methylation of
uridine to m3U1498, has no significant difference in growth rate
compared to wild type; however, mutant cell competition with wild
type showed an exponential decrease in the mutant cells as the
number of growth cycles increased.22 The doubling time of the
methyltransferase RsmF (responsible for m5C1407) knockout strain
is increased by 23% compared to wild type in rich media and 48% in
minimal media at 42 °C.21 The doubling time of the double knockout
strain for loss of methyltransferases RsmH and RsmI, which methyl-
ate the N4 and 20-O-positions of C1402, respectively, is increased by
29% over wild type.23 The luciferase reporter assay illustrated that
lack of methylation at C1402 also affected non-AUG initiation and
fidelity of translation.23 Our data support these findings in which
methylation in the decoding region is important, although not
essential, for cell growth, and suggests subtle effects in maintaining
key tertiary interactions for proper ribosomal function.
Figure 3. The circular dichroism spectra of the decoding region analogues (panel A:
unmodified C-C-U, singly modified m4Cm-C-U, and fully modified m4Cm-m5C-m3U
(wild type); panel B: decoding regions from E. coli, C. acetobutylicum and T.
thermophilus) are shown. Each curve represents the average of five scans taken at
room temperature.
were carried out. The spectrum of the unmodified decoding region
RNA (C-C-U) has a peak maximum at 266 nm and peak minimum
at 235 nm (Fig. 3A). The spectra for fully modified (m4Cm-m5C-
m3U) and singly modified m4Cm decoding region analogues have
similar peak maxima and minima. The CD spectra show that all
of the analogues have typical A-form RNA conformations.39 Com-
parison of CD spectra for the three bacterial decoding region ana-
logues (E. coli, C. acetobutylicum and T. thermophilus) reveals that
these related RNAs also share similar structural features (Fig. 3B).
The spectrum of the T. thermophilus RNA has an additional shoulder
between 275 and 285 nm, suggestive of an alternate conformation
or unique stacking arrangement with the m5C residues. With the
exception of the T. thermophilus analogue, there are no significant
effects of modified nucleotides on the overall conformations of
the decoding region analogues. These results are consistent with
the thermal melting data in which the modified nucleotides have
only subtle effects on stability, and the stabilizing and destabilizing
effects are compensatory, such that the overall stabilities are chan-
ged very little. Similarly, the CD data suggest that modified nucle-
otides in the decoding region do not change the average
conformations of the RNA.
3. Conclusions
In order to incorporate modified nucleotides site specifi-
cally into bacterial decoding region model systems, an N4,20-O-
dimethylcytidine (m4Cm) phosphoramidite was synthesized from

2724
S. K. Mahto, C. S. Chow / Bioorg. Med. Chem. 21 (2013) 2720–2726
the m4Cm nucleoside in 60% overall yield. A series of nine different
oligonucleotide strands containing the m4Cm, m5C, m3U, and Cm
modified nucleotides were generated by 50-BzH-20-ACE solid-phase
(254 lL, 1.8 mmol, 1 equiv to BzH-Cl) to another round-bottom
flask. One millilitre of solution B was added dropwise to solution
A, and the mixture was stirred for 10 min. Every 15 min, 1 mL of
solution B was added dropwise to solution A until solution B was
empty. Reaction progress was monitored by TLC. The reaction
was quenched with 5% NaHCO3 and extracted with CH2Cl2. The or-
ganic layer was washed with brine. The organic layer was dried
over Na2SO4, filtered, and purified on silica gel using 0.5% triethyl-
amine and 30–60% acetone in a hexane solvent gradient to afford 1
in 80% yield (790 mg). 1H NMR (500 MHz, CDCl3) d (ppm) 0.01–
0.03 (m, 18H), 2.84 (s, 3H), 3.62 (s, 3H), 3.80–4.00 (m, 2H), 4.05–
4.17 (m, 3H), 5.13 (d, 1H), 5.90–5.99 (m, 2H), 7.16–7.33 (m, 10H)
7.77 (d, 1H); 13C NMR (125 MHz, CDCl3) d (ppm) 1.61, 1.68, 1.72,
8.83, 28.04, 31.63, 36.70, 46.06, 58.88, 60.99, 61.14, 67.59, 67.79,
83.59, 84.20, 87.85, 94.90, 126.56, 126.64, 126.70, 127.26, 127.40,
127.58, 128.35, 128.49, 139.54, 144.20, 156.21, 164.44; HRMS
calcd for C30H45N3O8NaSi3 682.2412, found 682.2407.
synthesis on a lmol scale. The various oligonucleotide strands hav-
ing site-specifically modified nucleotides were employed in the
generation of a number of different bacterial decoding region mod-
el systems, which enabled biophysical studies to be carried out.
The thermal melting and CD studies provide insight into the struc-
tural role of the modified nucleotides. The m4Cm stabilizing effect
at position 1402 of the decoding region might play a role in stabi-
lizing interactions with helix 45 and mRNA during translation
through enhanced base stacking, as shown in crystal structures.
In contrast, the nucleotide m5C at position 1407 is part of the A-site
dinucleotide bulge where two adenine residues at positions 1492
and 1493 flip out during tRNA recognition. Therefore, it is not sur-
prising that m5C1407 has a destabilizing effect, which may facili-
tate dynamics of the decoding region or interactions with key
components of the translational machinery. Future experimenta-
tion, such as NMR or single-molecule spectroscopy, would be nec-
essary to verify such a mechanism.
4.1.2. 50-O-Benzhydryloxy-bis(trimethylsilyloxy)silyl-30-O-(N,N-
diisopropylamino)methoxyphosphonyl-N4,20-O-
These results are consistent with earlier studies showing that
modified nucleotides each play unique roles in regulating local
RNA stability, structure, or dynamics. Multiple modifications may
have additive effects on RNA conformation or stability, and the
overall effects may be subtle. Furthermore, the same modified
nucleotide at different or multiple locations within the same RNA
sequence may have additional stabilizing roles, such as m5C in
the T. thermophilus decoding region. The importance of using syn-
thetic approaches combined with biophysical approaches is dem-
onstrated in this study. Future studies such as single molecule
analysis on the different RNA constructs may allow more informa-
tion regarding the individual roles of modified nucleotides to be
gleaned. Ligand binding studies may also reveal whether the mod-
ified nucleotides influence important interactions with antibiotics
in the decoding region.
dimethylcytidine (2)
Compound 1 (330 mg, 0.5 mmol, 1.0 equiv) was added to a
round-bottom flask and dissolved in 2 mL of distilled CH2Cl2, and
the clear solution was stirred for a few minutes. Bis(N,N-diisopro-
pylamino)methoxyphosphine
(400
l
L,
367 mg,
1.4 mmol,
2.8 equiv) and 1-H-tetrazole (18 mg, 0.25 mmol, 0.5 eq) were
added to the clear solution while stirring, and the reaction pro-
ceeded for 12 h under argon. The reaction was quenched with
300 lL ethanol. The reaction was dried on rotary evaporator. The
crude product was purified using a hexanes/acetone/triethylamine
(4:1:0.5%) to hexanes/acetone/triethylamine (2:1:0.5%) solvent
gradient to yield 2 as a clear oil in 75% yield (307 mg). 1H NMR
(500 MHz, CDCl3) d (ppm) 0.01–0.03 (m, 18H), 1.00–1.16 (m,
12H), 2.50, 2.52, 2.54, 2.56 (each as a s, 6H), 3.62 (s, 3H), 3.80–
4.00 (m, 2H), 4.05–4.17 (m, 3H), 5.13 (d, 1H), 5.90–5.99 (m, 2H),
7.16–7.33 (m, 10H) 7.77 (d, 1H); 13C NMR (125 MHz, CDCl3) d
(ppm) 1.72, 1.74, 1.78, 8.21, 19.24, 19.31, 24.84, 24.89, 24.95,
25.03, 34.68, 43.01, 43.20, 43.25, 43.35, 46.86, 58.80, 83.96,
87.91, 89.03, 126.61, 126.69, 127.41, 128.47, 128.51, 128.57,
139.91, 144.26, 156.25, 164.48; 31P NMR (170 MHz, CDCl3) d
(ppm) 150.74, 151.87; HRMS calcd for C37H61N4O9NaPSi3
843.3382, found 843.3371 (Supplementary Figs. S1–S4).
4. Materials and methods
All reactions were carried out in anhydrous conditions under ar-
gon atmosphere at room temperature, unless noted otherwise. The
1H, 13C, and 31P NMR spectra were recorded on a Varian 400 MHz
or Unity 500 MHz spectrometer. ESI mass spectrometry was done
on a Waters Micromass Zq instrument. MALDI-TOF spectra was ob-
tained on a Bruker Ultraflex MALDI-TOF mass spectrometer. LC–MS
was carried out on a Waters LCT Premier XE. Flash column chroma-
tography was carried out on silica gel 60 (240–400 mesh). All NMR
solvents were from Cambridge Isotope Laboratories, Inc. Tetrazole
was purchased from Glen Research. Benzhydryloxy-bis(trimethyl-
siloxy)chlorosilane (BzH-Cl) was purchased from Dharmacon Re-
search, Inc. (Lafayette, CO). Methylene chloride (CH2Cl2) was
purchased from Fisher and distilled over CaH2. All other chemicals
were purchased from Sigma–Aldrich and Fisher and used without
further purification. The nucleoside m4Cm was prepared as de-
scribed previously.26
4.1.3. Synthesis of the decoding region RNAs
The m4Cm phosphoramidite was sent to Dharmacon Research
Inc. (Lafayette, CO) where the oligonucleotide synthesis was car-
ried out. The bacterial decoding region RNAs (Table S1, Supplemen-
tary data) were each synthesized on a 1 lmol scale.
4.2. RNA deprotection
The removal of protecting groups on the phosphate backbone
and exocyclic amines was carried out immediately after synthesis
of the oligonucleotide. The methyl protecting groups on the phos-
phate backbone were removed by using disodium 2-carbamoyl-2-
cyanoethylene 1,1-dithiolate (S2Na2) while the RNA was still on the
polystyrene support.42 Next, 40% methylamine in water43,44 was
used for deprotection of the exocyclic amines. This reaction also re-
moved the acetyl protection of the 20-bis-acetoxyethyl orthoester
to generate 20-bis-hydroxyethyl orthoester and cleaved the oligo-
nucleotide from the solid support. These steps were done at
Dharmacon Research. The RNAs were obtained as the 20-bis-
hydroxyethyl orthoester protected forms. The 20-orthoester was
4.1. Phosphoramidite synthesis
4.1.1. 50-O-Benzhydryloxy-bis(trimethylsilyloxy)silyl-N4,20-O-
dimethylcytidine (1)
In a round-bottom flask, m4Cm (408 mg, 1.5 mmol, 1 equiv) was
dissolved in 5 mL of anhydrous DMF. To prepare solution A, 5 mL of
distilled CH2Cl2 and 212 lL of diisopropylamine were added to the
solution at 0 °C. Solution B was prepared at 0 °C by adding
benzhydryloxy-bis(trimethylsiloxy)chlorosilane (BzH-Cl) (765 mg,
1.8 mmol, 1.2 equiv), 5 mL of CH2Cl2, and diisopropylamine
deprotected with 400
and pH adjusted to 3.8 with TEMED. The RNA was incubated with
lL of buffer containing 100 mM acetic acid

S. K. Mahto, C. S. Chow / Bioorg. Med. Chem. 21 (2013) 2720–2726
2725
buffer for 30–35 min at 60 °C. The sample was completely dried on
a Speedvac evaporator.
(1.0 ꢃ 104 cmꢁ1 Mꢁ1 at pH 7.0) and cytidine (7.6 ꢃ 103 cmꢁ1 Mꢁ1 at
pH7.0)wereusedinsteadof3-methyluridine(1.0 ꢃ 104 cmꢁ1 Mꢁ1 at
pH 7.0)46 and methylated cytidines (7.6 ꢃ 103 cmꢁ1 Mꢁ1 at pH 7.0)46
for all RNAs because the nearest-neighbor extinction coefficients for
methylatednucleotidesareunknown,thereforesomeerrormightexist
forthemodifiedRNAs.
4.3. RNA purification
The dried RNA samples were dissolved in double deionized
water (ddH2O). The UV absorbance values were obtained and the
concentrations were calculated by using the Beer–Lambert equa-
tion (Eq. 2):
4.5. Circular dichroism experiments
The circular dichroism (CD) spectra for all RNAs were obtained
on a Chirascan CD spectrometer equipped with a water bath to
control the temperature. Equimolar concentrations of the two
A ¼
in which
e
ꢂ C ꢂ k
ð2Þ
e, C and k are the extinction coefficient, concentration, and
path length, respectively. The extinction coefficients (e) are 190,400
strands (9.5
to 90 °C followed by slow cooling to 0 °C. The samples were then
diluted to 3.8 M in 1 mL of buffer. The CD spectra for the RNAs
were acquired from 220 to 320 nm in 1.0 nm steps with a band-
width of 1.0 nm at 25 °C. The samples were scanned five times
and an average was taken. The baseline subtraction and smoothing
using the Savitsky–Golay algorithm were performed for the ac-
quired averaged-CD spectra. The ellipticity values, h, for the 220–
lM, 400 lL total volume) were annealed by heating
and 242,500 cmꢁ1 Mꢁ1 for the 50 and 30 halves of the decoding re-
gion, respectively. Authenticity and purity of RNA samples were
confirmed by matrix-assisted laser desorption ionization-time of
flight mass spectrometry (MALDI-TOF MS) and polyacrylamide gel
electrophoresis (PAGE). The oligonucleotides of the decoding region
were purified by a using 20% denaturing PAGE (19:1 acrylamide/
bisacrylamide) in 0.5 M TBE buffer. Ethanol precipitations were
carried out after electroelution. The samples were dialyzed in
RNase-free, ddH2O for 3 days. RNA identities and purities were
reconfirmed by MALDI-TOF MS (Fig. S5, Supplementary data). The
incorporation of modified nucleotides was further confirmed by
P1 nuclease digestion or NaOH hydrolysis and phosphatase
digestion by bovine calf intestinal phosphatase (CIF) and analysis
of the resulting nucleosides by either high performance liquid
chromatography (HPLC) or liquid chromatography mass spectrom-
etry LC–MS. The LC–MS spectra of the 50 half of modified decoding
region (50-GCACAGACCG(m4Cm)CCGU(m5C)ACACC-30) showed
seven different peaks (Fig. S6, Supplementary data), corresponding
to six nucleosides (C, U, m5C, G, m4Cm and A) and dinucleotide
m4CmpC. The dinucleotide peak m4CmpC further confirmed that
m4Cm is 20-O-methylated, which leads to resistance against RNA
hydrolysis.
l
320 nm wavelength range were converted to
¼ h=32:98 ꢂ C ꢂ l
in which C and l denote the concentration of the sample and path
length of the cuvette, respectively.47 The data were plotted as
De using Eq 4:
D
e
ð4Þ
D
e
versus the wavelength. Exact RNA concentrations of the CD samples
were determined from the UV absorbance using the Beer–Lambert
equation. All CD experiments were done in triplicate.
Acknowledgments
We thank Drs. J. SantaLucia, A. Feig, B. Ksebati, L. Hryhorczuk,
and B. Shay for technical assistance and helpful discussions. This
work was supported by the National Institutes of Health
(AI061192, AI055496, and GM087596).
Supplementary data
4.4. UV-melting experiments
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.03.010.
In separate tubes, 2 OD units of purified, desalted RNA strands
were dissolved in 100 lL melting buffer (20 mM sodium cacodyl-
ate, 1 M NaCl and 0.5 mM Na2EDTA at pH 7.0). Each strand RNA
concentration was measured at 90 °C. Equimolar concentrations
were calculated for both strands X and Y by using Eq. 3.45
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