S-Geranyl-2-thiouridine wobble nucleosides of bacterial tRNAs; Chemical and enzymatic synthesis of S-geranylated-RNAs and their physicochemical characterization

Article abstract of DOI:10.1093/nar/gkw727

Recently, highly lipophilic S-geranylated derivatives of 5-methylaminomethyl-2-thiouridine (mnm5geS2U) and 5-carboxymethylaminomethyl-2-thiouridine (cmnm5geS2U) were found at the first (wobble) anticodon position in bacterial tRNAs specific for Lys, Glu and Gln. The function and cellular biogenesis of these unique tRNAs remain poorly understood. Here, we present one direct and two post-synthetic chemical routes for preparing model geS2U-RNAs. Our experimental data demonstrate that geS2U-RNAs are more lipophilic than their parent S2U-RNAs as well as non-modified U-RNAs. Thermodynamic studies revealed that the S-geranyl-2-thiouridine-containing RNA has higher affinity toward complementary RNA strand with G opposite the modified unit than with A. Recombinant tRNA selenouridine synthase (SelU) exhibits sulfur-specific geranylation activity toward model S2U-RNA, which is composed of the anticodon-stem-loop (ASL) from the human tRNA^Lys3 sequence. In addition, the presence of magnesium ions is required to achieve appreciable geranylation efficiencies.

Full text of DOI:10.1093/nar/gkw727

10986–10998 Nucleic Acids Research, 2016, Vol. 44, No. 22
Published online 26 August 2016
doi: 10.1093/nar/gkw727
S-Geranyl-2-thiouridine wobble nucleosides of
bacterial tRNAs; chemical and enzymatic synthesis of
S-geranylated-RNAs and their physicochemical
characterization
1
,†
2,†
2
Malgorzata Sierant , Grazyna Leszczynska , Klaudia Sadowska ,
2
1
2
1,*
Agnieszka Dziergowska , Michal Rozanski , Elzbieta Sochacka and Barbara Nawrot
1
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Bioorganic
2
Chemistry, Sienkiewicza 112, 90-363 Lodz, Poland and Institute of Organic Chemistry, Faculty of Chemistry, Lodz
University of Technology, Zeromskiego 116, 90-924 Lodz, Poland
Received June 29, 2016; Revised July 30, 2016; Accepted August 06, 2016
ABSTRACT
tional modifications important for their structure and func-
tion. According to available online databases of RNA mod-
ifications (http://rna-mdb.cas.albany.edu; http://modomics.
genesilico.pl) (1,2), 91 of the 109 currently known RNA nu-
cleoside modifications have been found in tRNA molecules.
The most frequently modified sites in tRNAs are nucleoside
34 (the anticodon wobble position) and the purine nucleo-
side 37 (adjacent to the anticodon). These modifications in-
crease the fidelity of codon recognition, allow for efficient
recognition of several codons by the same tRNA (3) and
prevent frame-shift reading. Modifications in other tRNA
domains mainly contribute to tRNA folding and stability
Recently, highly lipophilic S-geranylated derivatives
of 5-methylaminomethyl-2-thiouridine (mnm5geS2U)
and
5-carboxymethylaminomethyl-2-thiouridine
(cmnm5geS2U) were found at the first (wobble) an-
ticodon position in bacterial tRNAs specific for Lys,
Glu and Gln. The function and cellular biogenesis of
these unique tRNAs remain poorly understood. Here,
we present one direct and two post-synthetic chem-
ical routes for preparing model geS2U-RNAs. Our
experimental data demonstrate that geS2U-RNAs are
more lipophilic than their parent S2U-RNAs as well
as non-modified U-RNAs. Thermodynamic studies
revealed that the S-geranyl-2-thiouridine-containing
RNA has higher affinity toward complementary
RNA strand with G opposite the modified unit than
with A. Recombinant tRNA selenouridine synthase
(
4,5). Certain stress-induced changes in tRNA modification
patterns are postulated to function as another level of reg-
ulating gene expression (6).
At the wobble position, 5-substituted uridines (R5U) and
2
-thiouridines (R5S2U) are present in the tRNAs for ly-
Lys
Glu
sine (tRNA ), glutamic acid (tRNA ) and glutamine
Gln
(tRNA ). These modified bases promote the reading of
(
SelU) exhibits sulfur-specific geranylation activity
both NNA and NNG codons, tending to favor the recog-
nition of adenosine-containing codons (7,8). These mod-
ifications are essential for the viability of eukaryotic and
prokaryotic cells. The absence of a side chain in the C5 po-
sition or the lack of a sulfur atom in the C2 position leads
to detrimental frame-shift reading (9).
Most of the RNA modifications reported to-date in-
volve the introduction of small and hydrophilic groups.
However, several years ago an unusual, large hydropho-
bic group was found in bacterial tRNAs (10). This hy-
drophobic group was later identified as a geranyl group
toward model S2U-RNA, which is composed of the
anticodon-stem-loop (ASL) from the human tRNA
sequence. In addition, the presence of magnesium
ions is required to achieve appreciable geranylation
efficiencies.
Lys3
INTRODUCTION
A majority of the RNA species in cells undergo post-
transcriptional modifications, and the chemical groups in-
troduced by these modifications have distinct effects not
only at the site of modification but also on the global
structure of the modified RNA. In particular, transfer
RNAs (tRNAs) acquire chemically diverse posttranscrip-
(
11) and represented the first isoprenylated nucleoside ob-
served in natural ribonucleic acids. Previously, a similar
modification was found in proteins, where geranylgeranyl
group (introduced by geranyl transferases) facilitate at-
*
To whom correspondence should be addressed. Tel: +48 42 6803248; Fax: +48 42 6815483; Email: bnawrot@cbmm.lodz.pl
Equal contribution.
†
ꢀ
The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.
C
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which
permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact
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Nucleic Acids Research, 2016, Vol. 44, No. 22 10987
tachment to cellular membranes (similar to lipid anchors)
and is important for protein-protein interactions (12). The
geranyl group in tRNA is attached to the sulfur atom
in 5-methylaminomethyl-2-thiouridine (mnm5S2U) and
that an S2U-RNA homologous in sequence to the hu-
Lys3
man tRNA
anticodon stem-loop (ASL) domain is ger-
anylated by the recombinant SeIU enzyme, albeit only
2+
in the presence of Mg . The efficiency of enzymatic
S-geranylation of the S2U-RNA model (lacking the 5-
(c)mnm-substituent and trimmed to the ASL domain only)
and the demonstrated substrate tolerance suggest promis-
ing approach for future research based on the enzymatic
preparation of S-geranylated-ASL oligoribonucleotides.
The geranylated RNA models produced in this study were
characterized in terms of their lipophilicity and binding
preferences in comparison with their parent S2U-RNAs as
well as non-modified U-RNAs.
5
-carboxymethylaminomethyl-2-thiouridine (cmnm5S2U)
(Figure 1). The resulting S-geranylated-2-thiouridines
(
mnm5geS2U and cmnm5geS2U) account for up to 6.7% of
the (c)mnm5S2Us (∼400 geranylated nucleosides) within a
cell (11).
The bacterial tRNA selenouridine synthase (SelU) has
been recognized not only as the biocatalyst for the S-
geranylation of tRNAs in cells (9–11) but also as the cat-
alyst for S→Se replacement (13). Liu et al. (11) claimed
that the S-geranylation of tRNAs is an alternative to the
selenation of tRNA, with S-geranylation being executed
at low selenium concentrations. Thus, S→Se replacement
and S-geranylation are catalyzed by the same enzyme op-
erating on the same substrate (S2U). However, this dual
modus operandi leads to an awkward (and perhaps truly
conflicting) situation: for selenation, the sulfur atom must
be converted into a fairly good leaving group (the C2 atom
MATERIALS AND METHODS
Chemical synthesis of geS2U-RNA
Synthesis of S-geranyl-2-thiouridine phosphoramidite 1.
ꢁ
ꢁ
ꢁ
2 -O-(tert-Butyldimethylsilyl)-5 -O-(4,4 -dimethoxytrityl)-
ꢁ
S-geranyl-2-thiouridine (6). 5 -O-DMTr-geS2U (5) (14)
(650 mg, 0.93 mmol) was dissolved in anhydrous pyridine
(10 ml). Then imidazole (190 mg, 2.79 mmol, 3 equiv.) and
TBDMSCl (209 mg, 1.40 mmol, 1.5 equiv.) were added
and the mixture was stirred at room temperature (rt) for
20 h. The reaction was quenched with water (20 ml) and
−
to be attacked by an Se nucleophile). In contrast, dur-
ing S-geranylation, the sulfur atom (preferably within an
−
RS -like moiety) is expected to act as the nucleophile.
Recently, we proposed a ‘linear’ model wherein the S-
geranyl-2-thiouridine (geS2U) is not a final product but
is rather an intermediate in the S2U→Se2U conversion
the mixture was extracted with CHCl (3 × 30 ml). The
3
combined organic layers were dried over MgSO , filtered
4
(
14). This hypothesis was supported significantly by our
and concentrated under reduced pressure. The residue was
success in performing the two-step chemical transforma-
tion S2U→geS2U→Se2U and was further supported by
evidence from Jager et al. (9). Several mutations intro-
duced into the SelU (MnmH) polypeptide chain allowed
the amino acids responsible for interactions within the
tRNA molecule and for binding of modified nucleosides
co-evaporated with anhydrous toluene and purified on
a silica gel column (MeOH/CH Cl 4:96, v/v) to yield
2
2
ꢁ
a 2 -O-silylated isomer 6 as a white foam (302 mg, 0.37
mmol, 40%) and an undesired 3 -O-silylated isomer in
45%. To get additional quantities of 6, the 3 -isomer was
partially isomerized in methanol with a trace amount of
ꢁ
ꢁ
(
the Walker’s motif in the P-loop domain) to be identified.
In addition, two amino acids important for S-geranylation
G67) and selenation (C97) have been identified in the rho-
triethylamine (TEA) and additional 114 mg of 6 were
isolated rising the overall yield to 55%. TLC R = 0.75
f
(
(CH Cl /acetone 90:10, v/v). NMR (␦ [ppm], DMSO-d ):
2
2
6
1
danese domain.
H (250 MHz) 0.02 (s, 3H), 0.06 (s, 3H), 0.86 (s, 9H), 1.56 (s,
It has been suggested that geranyl hydrophobic group
confers unique physicochemical and conformational fea-
tures to the whole tRNA molecule. Such modified tRNA
3H), 1.63 (s, 3H), 1.71 (s, 3H), 1.98-2.00 (m, 2H), 2.03–2.06
(m, 2H), 3.25 (dd, J = 2.8 Hz, J = 11.2 Hz, 1H), 3.38 (dd,
J = 4.2 Hz, J = 11.2 Hz, 1H), 3.75 (s, 6H), 3.79–3.82 (m,
2H), 4.07–4.12 (m, 2H), 4.30 (t, J = 7.5 Hz, 1H), 5.04-5.06
(m, 1H), 5.29-5.32 (m, 1H), 5.38 (d, J = 7.5 Hz, 1H), 5.64
(d, J = 7.5 Hz,1H), 5.73 (d, J = 4.9 Hz, 1H), 6.91–7.38 (m,
ꢁ
ꢁ
ꢁ
ꢁ
favorably recognizes 5 -NNG-3 codons over 5 -NNA-3
codons (11). This enhancement in specificity was recently
reported by Wang et al., who found that the thermal sta-
bility of a DNA duplex containing a geS2T-dG base pair
was higher than a duplex containing a geS2T-dA base pair
13
13H), 7.90 (d, J = 7.5 Hz, 1H); C (176 MHz) -4.76, -4.22,
16.59, 18.01, 18.32, 25.92, 26.08, 26.37, 29.93, 39.05, 55.56,
63.24, 70.16, 76.88, 84.85, 86.67, 91.77, 109.35, 113.81,
117.63, 124.09, 127.39, 128.08, 128.39, 130.24, 131.52,
135.31, 135.54, 139.23, 141.79, 145.04, 158.69, 162.04,
(
15). The enhanced discrimination between G- and A-ended
codons conferred by S-geranylation has been attributed to
the conversion of the N3H donor center into an N3 accep-
tor center.
+
166.67. HRMS (FAB) calc. for C H N O SSi [M+H]
4
6
60
2
7
Although the S-geranylation of tRNAs has been found
in several bacterial strains (e.g. Escherichia coli, Enterobac-
ter aerogenes, Pseudomonas aeruginosa and Salmonella Ty-
phimurium), detailed data on the properties and cellular
functions of these tRNAs have not been reported, with
the exception of data on the hydrophobicity and structural
characteristics of mnm5geS2U and cmnm5geS2U at the nu-
cleoside level (16).
813.3969, found 813.3999.
ꢁ
ꢁ
ꢁ
ꢁ
2 -O-(tert-Butyldimethylsilyl)-5 -O-(4,4 -
dimethoxytrityl)-S-geranyl-2-thiouridine-3 -O-(cyanoethyl-
N,N-diisopropyl)phosphoramidite (1). To a solution of
ꢁ
ꢁ
5 -O-DMTr-2 -O-TBDMS-S-geranyl-2-thiouridine 6 (256
mg, 0.32 mmol) in anhydrous THF (3 ml, under argon
atmosphere), 4-dimethylaminopyridine (DMAP) (7.7 mg,
0.06 mmol, 0.2 equiv.), N,N-diisopropylethylamine (DIEA)
(221 ␮l, 1.27 mmol, 4 equiv.) and 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite (141 ␮l, 0.63 mmol, 2
equiv.) were added. The reaction mixture (protected from
Here, we report three original and effective methods
for the chemical synthesis of model RNA oligonucleotides
containing S-geranyl-2-thiouridine. We also demonstrate

10988 Nucleic Acids Research, 2016, Vol. 44, No. 22
Figure 1. Chemical structures of S-geranylated nucleosides.
the daylight) was stirred at rt. After reaction completion
␮l). The combined washings were evaporated on a Speed-
(
(
2 h, TLC control) the mixture was diluted with AcOEt
30 ml), treated with 5% aq. NaHCO3 (10 ml) and washed
Vac concentrator and the solid residue was treated with
.
Et3N 3HF in NMP (1:1 v/v, 120 ␮l) for 24 h at rt (the check-
with water (10 ml). The combined organic layers were dried
over MgSO4, filtered and concentrated under reduced
pressure. The oil residue was co-evaporated with anhydrous
toluene and purified on a silica gel flash column (petroleum
ether/AcOEt 1:1, v/v) to yield 1 as a white foam (214 mg,
point B). The reaction was quenched by addition of 240 ␮l
of ethoxytrimethylsilane and the crude RNA was precipi-
tated using 600 ␮l of tert-butyl methyl ether. The fully de-
protected RNA was purified by anion-exchange (IE) HPLC
(Source 15Q 4.6/100PE) at a constant flow rate of 1 ml/min.
The column was eluted with a linear gradient 50–500 mM
NaBr in a 20 mM Na2HPO4–NaH2PO4 buffer solution pH
7.5, containing 50 ␮M EDTA and 10% ACN. Fractions
containing the desired product were collected, concentrated
and desalted on a C-18 cartridge (Sep-Pak, Waters). The
desalted RNA was lyophilized (19 OD260 units, ca. 55%
yield) and analyzed by MALDI-TOF mass spectrometry
(m/z 5481, MW 5483) (Supplementary Figure S1).
0.21 mmol, 66%). TLC: Rf = 0.83 (CH2Cl2/AcOEt/TEA,
1
1
0:10:1, v/v). NMR (␦ [ppm], C6D6): H (250 MHz)
-
0.07–0.17 (m, 6H), 0.66–0.96 (m, 7H), 0.99–1.01 (m, 9H),
1
2
4
6
7
1
.13–1.19 (m, 5H), 1.54–1.67 (m, 10H), 1.90–2.08 (m, 5H),
.98–3.77 (m, 12H), 3.86–3.94 (m, 2H), 4.10-4.51 (m, 2H),
.65–4.77 (m, 1H), 5.30–5.40 (m, 1H), 5.97–6.05 (m, 1H),
.19–6.27 (m, 1H), 6.76–6.82 (m, 4H), 7.05–7.08 (m, 1H),
.18–7.60 (m, 8H), 8.03–8.13 (m, 1H); 31P (101 MHz)
49.07, 151.91; HRMS (ESI): calc. for C55H77N4O8PSSi
+
[
M+H] 1013.5047, found 1013.5042.
Chemical synthesis of geS2U-RNA via the post-synthetic ger-
anylation of support-linked S2U-RNA (method II). The
ꢁ
ꢁ
Chemical synthesis of geS2U-RNA via direct incorporation
of the geS2U unit (method I). A geS2U–RNA oligomer of
S2U–RNA of the 5 -GUUGACUS2UUUAAUCAAC-3
sequence was synthesized automatically on a 0.2 ␮mole
scale analogously to the synthesis of geS2U–RNA with the
exception of the use of S2U-phosphoramidite 2 (17), and
conditions for the oxidation steps, where a solution of t-
BuOOH in toluene (0.25 M, 8 equiv.) was applied for 2 min.
The support carrying detritylated S2U-RNA was worked-
up until the checkpoint A (vide supra) was reached. Now the
sample was treated with a mixture of geranyl bromide (4.2
␮l, 213 equiv.), triethylamine (3 ␮l, 213 equiv.) and ethanol
(45 ␮l) and the suspension was shaken vigorously for 3 h
at rt. The supernatant was removed and the support was
washed with anhydrous ethanol (3 × 150 ␮l) and dried in
vacuo for 30 min, followed by treatment with ethanolic am-
monia (8 M, 340 ␮l) for 8 h at rt. The supernatant was re-
moved and the support was washed with ethanol (3 × 150
␮l). The combined supernatant and washings were evapo-
rated on a Speed-Vac concentrator. To the solid residue a
solution of Et3N·3HF in NMP (1:1, v/v, 120 ␮l) was added
and the reaction was left for 24 h at rt. Further RNA isola-
tion and purification was analogous to the procedure after
the checkpoint B (vide supra) and 13 OD260 units of geS2U-
RNA were obtained (ca. 40% yield). Taking into account
that the average yield of the synthesis of S2U-RNA is 71%
(24 OD from 0.2 ␮mole scale), the efficacy of the CPG-S2U-
RNA → CPG-geS2U-RNA conversion could be estimated
at 55%.
ꢁ
ꢁ
the 5 -GUUGACUgeS2UUUAAUCAAC-3 sequence was
synthesized automatically on a 0.2 ␮mole scale with the
use of an H6 GeneWorld DNA/RNA automated synthe-
sizer (K&A, Laborgeraete GbR, Schaafheim, Germany).
The commercially available phosphoramidites of A, C, U
ꢁ
ꢁ
and G protected on the 5 - and 2 -hydroxy functions with
DMTr and TBDMS groups, respectively, were used. The
exocyclic amine functions were masked with phenoxyacetyl
(
A and G units), or acetyl (C) groups (Proligo). The rC(tac)-
succinyl-CPG (Proligo) support and 0.07 M acetonitrile
ACN) solutions of the monomers were used. The geranyl-
(
modified S2U-phosphoramidite 1 was dissolved in a mix-
ture of THF/ACN (1:9, v/v). All amidities were used in a
1
0-fold molar excess and the coupling in the presence of
a BMT activator (0.25 M in ACN) was executed for 10
min. For the oxidation step, a solution of I2 (0.02 M in
THF/H2O/pyridine 90.54:9.05:0.41, v/v/v) was used for
5
min. Capping was performed with TAC2O in THF us-
ing a mixture of Fast Deprotection Cap A (Proligo) and
Cap B (Proligo) (1.1:1, v/v) for 2 min. After the last cou-
pling the DMTr group was removed and the support was
washed, dried and transferred to a screw cap glass vial.
Then a TEA/ACN mixture (265 ␮l, 1:1, v/v) was added and
the suspension was stirred for 20 min, and then the volatile
components were removed. The support-bound RNA was
washed with ACN (3 × 100 ␮l), dried in vacuo for 30 min
The selectivity of the post-synthetic geranylation of S2U–
RNA was confirmed by enzymatic hydrolysis of the final
RNA with nuclease P1 and alkaline phosphatase (18,19).
The resulting mixture of nucleosides was analyzed by RP-
(
a checkpoint A), and treated with ethanolic ammonia (8
M, 340 ␮l) at rt for 8 h. The supernatant was removed and
the support was washed with anhydrous ethanol (3 × 150

Nucleic Acids Research, 2016, Vol. 44, No. 22 10989
HPLC on a C18 column (Ascentis, 4.6 × 250 mm) using
a mixture of buffer A (0.1 M CH3COONH4) and buffer
B (40% ACN in 0.1 M CH3COONH4) at a constant flow
rate of 1 ml/min (details are given in Supplementary Figure
S2A). The retention time (Rt) of the geS2U was compared
to the Rt of the reference samples in a separate control ex-
periment (Supplementary Figure S2B).
centrifugation for 0.5 h at 15 000 ×g, the clarified extract
was loaded on a column with ∼3 ml of HisPur Cobalt
Resin (ThermoScientific) pre-equilibrated in a buffer A
(50 mM sodium phosphate, pH 8.0, 300 mM NaCl and 10
mM imidazole). After washing with 30 ml of buffer A, the
protein was eluted with 20 ml buffer A containing 100 mM
imidazole, followed by two consecutive dialyses against 1 l
of a 50 mM sodium phosphate buffer, pH 8.0. Purification
of the dialyzed protein was repeated by affinity chromatog-
raphy and the solution of the protein was dialyzed twice
against 1 l of 20 mM Tris HCl, pH 8.0. After dialysis,
the protein was concentrated (using an Amicon Ultra
centrifugal filter unit 30K, Merck–Millipore), and the final
concentration was determined spectrophotometrically. The
protein was preferably used immediately after purification.
The surplus protein was frozen in liquid nitrogen and
Chemical synthesis of geS2U–RNA via the post-synthetic
geranylation of fully deprotected S2U-RNA (method III).
The S2U–RNA was synthesized automatically in a 0.2
␮
mole scale according to the procedure described above.
The support carrying detritylated S2U–RNA was treated
◦
with aq. NH3/EtOH (3 : 1, v/v, 1.5 ml) for 16 h at 40 C. The
supernatant was collected and the support was washed with
ethanol/H2O (1 : 1, v/v, 3 × 150 ␮l). The combined frac-
tions were evaporated on a Speed-Vac concentrator. To the
solid residue a solution of Et3N·3HF in NMP (1:1 v/v, 120
◦
stored at −70 C.
␮
l) was added and the reaction mixture was left for 24 h at
Synthesis of geS2U–RNA by enzymatic geranylation with
rt. Further isolation and purification steps were performed
analogously as described in Method II. The desalted S2U–
RNA was lyophilized and analyzed by mass spectrometry
recombinant SelU enzyme. The 17-mer S2U-RNA (as-
h
ꢁ
ꢁ
signed as S ) of the 5 -UCAGACUS2UUUAAUCUGA-3
sequence, isosequential to the anticodon-stem-loop (ASL)
Lys3
(MALDI-TOF, m/z 5342, MW 5349). For the geranylation,
of human tRNA UUU (1 OD, 40 ␮g) was incubated with
to a solution of S2U-RNA (24 OD) in an EtOH/H2O mix-
ture (1/1, v/v, 90 ␮l), TEA (6 ␮l, 213 equiv.) and geranyl
bromide (8.4 ␮l, 213 equiv.) were added. The mixture was
shaken vigorously for 3 h at rt. The resultant solution was
diluted with 20% aq. EtOH and the RNA oligonucleotide
was isolated using a NAP-25 column (GE Healthcare). The
eluate was evaporated in a Speed-Vac concentrator and pu-
rified by IE HPLC according to the procedure described in
method I to furnish the geS2U–RNA product in 67% yield.
The relevant MALDI-TOF mass spectrum is shown in Sup-
plementary Figure S3.
the purified enzyme SelU-His6 (10 ␮g) in the presence of
5-fold molar excess of ammonium geranyl pyrophosphate
(Sigma-Aldrich). The reaction was performed in the buffer
composed of 10 mM Tris-HCl pH 8.0, 0.2 mM dithiothre-
itol and 2% glycerol without or with addition of MgSO4
salt (up to 100 mM), in total reaction volume 50 ␮l, at
◦
25 C. A reaction with the thermally inactivated enzyme
served as the negative control. After 24 h incubation the
mixture was analyzed by RP-HPLC (a Prominence chro-
matograph, Shimadzu, Japan) using a Kinetex 5␮ C-18 col-
˚
umn (100A; 250 × 4.60 mm, Phenomenex) eluted with a
mixture of two buffers (A: 0.1 M CH3COONH4; B: 0.1 M
CH3COONH4/40% ACN) according to the following gra-
dient: 0% B for 20 min.; 0→100 % B over 30 min.; then iso-
cratically 100% B for 10 min. The fractions collected at Rt
= 39 min were desalted using a standard SepPak C-18 (Wa-
ters) procedure and the identity of the geranylated product
was confirmed by MALDI-TOF MS (m/z 5477, MW 5483,
Supplementary Figure S4).
Enzymatic geranylation of S2U–RNA with a recombinant
SelU enzyme
Overexpression and purification of the SelU enzyme.
A
1
2
095 bp gene for selenouridine tRNA synthase (SelU, EC:
.9.1- EAL2 c13120) was amplified from total bacterial
RNA (E. coli, TOP10 strain) using a Transcriptor One-Step
RT-PCR kit (Roche) with the appropriate forward (Fow)
ꢁ
and reverse (Rev) primers: selU-For (5 -AAAAAAccat
Physicochemical characterization
ꢁ
ggATGCAAGAGAGACACACGGA-3 ) and selU-Rev
5 -ATATATctcgagCCGCGCCTTAACCCATTCC-3 ).
An insert coding the selU gene was cloned into a pET28c
expression vector (Novagen) between the T7 promoter
and the region encoding a His6 affinity tag. The recom-
binant protein was overexpressed in BL21 Star (DE3)
cells (Invitrogen) in 3 l of 2xYT medium supplemented
ꢁ
ꢁ
(
UV melting temperature measurements. All UV absorp-
tion measurements were carried out in a 1-cm path length
cells using a Cintra 4040 spectrophotometer, equipped with
a Peltier Thermocell (GBC, Dandenong, Australia), with
a detector set at 260 nm. Complementary RNA/RNA
or RNA/DNA oligonucleotide strands (detailed sequences
are listed in Table 1) were mixed in a phosphate buffer (10
mM sodium phosphate pH 7.4 containing 100 mM NaCl)
at the final 2 ␮M concentration. For the experiments with
the Ag+ ions mediated base-pairing three equivalents of
AgNO3 (6 ␮M final concentration) were added. The sam-
◦
with kanamycin (50 ␮g/ml). After growth at 37 C to an
OD600 = 0.6, the SelU synthesis was induced by addition
of isopropyl-␤-D-thiogalactopyranoside (1 mM) and a
◦
culture was continued to grow at 20 C for 4 h. After that,
the cells were harvested by centrifugation and the cell pellet
◦
◦
(
∼15 g of the wet mass) was resuspended in 20 ml of a 50
ples were then heated to 85 C, and cooled to 15 C with
◦
mM sodium phosphate buffer, pH 8.0, with the EDTA-free
Protease Inhibitor cocktail (Roche) and protamine sulfate
a temperature gradient of 1.5 C/min. The melting profiles
◦
were recorded from 15 to 85 C, with the temperature gradi-
◦
(
0.5 mg/ml) (Sigma-Aldrich). Cells were disrupted by son-
ication. All subsequent steps were performed at 4 C. After
ent of 0.5 C/min. The melting temperatures were calculated
◦
using the first derivative method implemented in the Cintra

10990 Nucleic Acids Research, 2016, Vol. 44, No. 22
Table 1. UV melting temperatures and Gibbs free energy values for RNA/RNA and RNA/DNA duplexes → ss RNA/DNA transitions
Tm after addition of
◦
+
◦
◦
No.
Name
Sequence
Tm ( C)
Ag ( C)
ꢀG (kcal/mol)
ꢁ
ꢁ
1
2
3
4
5
6
7
8
9
1
1
1
.
U/A
5 -GUUGACU U UUAAUCAAC-3
55.9 ± 0.3
59.5 ± 0.2
46.9 ± 1.0
53.5 ± 0.3
51.6 ± 0.5
50.1 ± 0.2
43.4 ± 0.4
46.6 ± 0.2
44.3 ± 0.4
44.1 ± 0.5
44.7 ± 0.2
43.3 ± 0.8
55.7 ± 0.1
− 15.1 ± 0.1
− 15.8 ± 0.1
− 10.7 ± 0.2
− 13.9 ± 0.3
− 12.9 ± 0.2
− 12.2 ± 0.2
− 9.4 ± 0.1
− 11.1 ± 0.1
− 10.1 ± 0.2
− 9.7 ± 0.1
− 9.8 ± 0.1
− 9.3 ± 0.1
ꢁ
ꢁ
ꢁ
3
- CAACUGA A AAUUAGUUG-5
ꢁ
ꢁ
.
S/A
5 -GUUGACU S UUAAUCAAC-3
58.7 ± 0.2
49.9 ± 0.3
53.5 ± 0.1
53.7 ± 0.5
50.0 ± 1.0
44.3 ± 0.1
44.8 ± 0.2
45.1 ± 1.7
47.4 ± 0.1
43.4 ± 0.2
45.1 ± 0.2
ꢁ
3
- CAACUGA A AAUUAGUUG-5
ꢁ
ꢁ
ꢁ
.
geS/A
U/G
5 -GUUGACU geS UUAAUCAAC-3
ꢁ
ꢁ
ꢁ
ꢁ
3
-CAACUGA A AAUUAGUUG-5
.
5 -GUUGACU U UUAAUCAAC-3
ꢁ
ꢁ
3
- CAACUGA G AAUUAGUUG-5
ꢁ
ꢁ
.
S/G
5 -GUUGACU S UUAAUCAAC-3
ꢁ
ꢁ
3
-CAACUGA G AAUUAGUUG-5
ꢁ
.
geS/G
U/dA
S/dA
geS/dA
U/dG
S/dG
geS/dG
5 -GUUGACU geS UUAAUCAAC-3
ꢁ
ꢁ
ꢁ
-CAACUGA G AAUUAGUUG-5
3
ꢁ
.
5 - GUUGACU U UUAAUCAAC-3
ꢁ
ꢁ
ꢁ
3
-d(CAACTGA dA AATTAGTTG)-5
ꢁ
ꢁ
.
5 - GUUGACU S UUAAUCAAC-3
ꢁ
3
-d(CAACTGA dA AATTAGTTG)-5
ꢁ
ꢁ
ꢁ
.
5 - GUUGACU geS UUAAUCAAC-3
ꢁ
3
-d(CAACTGA dA AATTAGTTG)-5
ꢁ
ꢁ
0.
1.
2.
5 - GUUGACU U UUAAUCAAC-3
ꢁ
ꢁ
ꢁ
3
-d(CAACTGA dG AATTAGTTG)-5
ꢁ
ꢁ
5 - GUUGACU S UUAAUCAAC-3
ꢁ
3
-d(CAACTGA dG AATTAGTTG)-5
ꢁ
ꢁ
5 - GUUGACU geS UUAAUCAAC-3
ꢁ
ꢁ
-d(CAACTGA dG AATTAGTTG)-5
3
Duplex abbreviated names and their sequences are given in columns 2 and 3. Tm values were determined by calculating the first derivative of the melting
curve functions generated by the melting software Cintra 4040. Duplexes (2 ␮M) were dissolved in 10 mM sodium phosphate (pH 7.4) containing 100 mM
NaCl. For sealing measurements, duplexes were incubated with 3 equiv. of AgNO3.
4
040 software. The calculation of thermodynamic parame-
ous and organic phases were separated by centrifugation
(10 min, 15 000 xg, rt). Approximately 900 ␮l aliquots of
the aqueous and 1-octanol layers were taken, and the con-
centrations of dissolved oligonucleotides were assessed by
UV absorption measurements at 260 nm (Cintra 4040 spec-
trophotometer). The partition coefficient P, defined as the
ratio of the oligonucleotide concentrations in the organic
and aqueous phases, was determined and its log P-value
was calculated according to the equation: Log Po/w = Log
◦
◦
◦
ters (ꢀG , ꢀH and ꢀS ) was done by numerical fitting of a
given melting curve using a two-state model algorithm pro-
vided by a MeltWin v.3.5 software. Each result was taken as
an averaged one from three independent experiments.
Circular Dichroism Measurements. Circular dichroism
measurements were performed using a J-815 circular
dichroism (CD) spectrometer (Jasco, Japan). The duplex
samples were prepared at a 2 ␮M concentration in a 10 mM
sodium phosphate buffer, pH 7.4 containing 100 mM NaCl.
Equimolar amounts of complementary oligonucleotides
[c
RNA o
] − Log [cRNA]w.
◦
were mixed in a buffer, heated to 95 C and slowly cooled
RESULTS
down to room temperature. CD spectra were obtained us-
ing a 0.5 cm path length quartz cell (Helma). The recording
parameters were: scan speed 50 nm/min, response time 2 s,
band width 1.0 nm and step resolution 1.0 nm. The spec-
Design of model RNAs
We designed a model 17-mer geS2U-RNA oligomer
ꢁ
(abbreviated as geS) with the following sequence: 5 -
◦
ꢁ
tra were recorded at 24 C, in the wavelength range from
GUUGACUgeS2UUUAAUCAAC-3 . This sequence is
2
00 to 360 nm. The spectrum recorded for the buffer was
numerically subtracted from the spectrum for each sample
recorded in triplicate), and the resultant averaged spectra
homologous to the sequence of the ASL of E. coli
Lys
tRNA UUU. For the enzymatic geranylation, an anal-
(
ogous ASL model of S2U–RNA with the sequence
ꢁ
ꢁ
were smoothed with a Savitzky–Golay algorithm (convolu-
tion width 7).
5 -UCAGACUS2UUUAAUCUGA-3 was used; this se-
quence is identical to that of the anticodon-stem-loop of
Lys3
human tRNA UUU. The S2U–RNA was abbreviated as
h
h
Determination of Log P of geS2U–RNA. The partition co-
efficient (P) was determined in a water/1-octanol immisci-
ble bi-phase system. The samples of single stranded RNAs
S , while the geranylated product was abbreviated as geS .
The modified unit in both RNA models was inserted in the
location corresponding to the wobble position in the intact
tRNAs. For UV melting experiments, CD measurements
and lipophilicity measurements, the corresponding analogs
of geS were prepared, namely U–RNA (abbreviated as U)
and S2U–RNA (abbreviated as S) as well as their comple-
mentary strands with A or G nucleotides (abbreviated as A
or G, respectively) located opposite of the modified unit.
(
1 OD260 unit of U, S or geS) were suspended in a mix-
ture of 1 ml of RNase free water and 1 ml of 1-octanol
reciprocally saturated with 1-octanol or RNase free water,
respectively. The samples were shaken vigorously at room
temperature for at least 2 h to ensure that the partitioning
of the oligonucleotide reached the equilibrium. The aque-

Nucleic Acids Research, 2016, Vol. 44, No. 22 10991
Chemical synthesis of geS2U–RNA
for automated solid phase synthesis. A 10-fold molar excess
of each amidite was used, and coupling reactions were con-
ducted for 10 min using BMT in ACN as an activator. Oxi-
dation was carried out with an I2/THF-H2O-pyridine solu-
tion. After chain assembly was complete, the DMTr group
was removed, and CPG-bound RNA was treated with TEA
in ACN to remove the 2-cyanoethyl groups from the tri-
ester phosphate residues. Upon treatment with anhydrous 8
M ethanolic ammonia (8 h, rt) the oligoribonucleotide was
released from the CPG-support, and the remaining base-
Chemical syntheses of model oligoribonucleotides contain-
ing S-geranyl-2-thiouridine were performed using three dif-
ferent chemical methods, numbered I-III (Scheme 1). These
approaches included direct incorporation of the geS2U unit
into the RNA chain by standard phosphoramidite chem-
istry using the monomer 1 (I), the post-synthetic gerany-
lation of CPG-linked S2U–RNA (II) or the post-synthetic
geranylation of fully deprotected S2U–RNA (III).
ꢁ
labile groups were removed. Finally, 2 -OH groups were de-
Optimization of conditions of the geS2U-RNA synthe-
sis. To determine an effective protocol for the synthe-
sis of geS2U-containing oligoribonucleotides via phospho-
ramidite chemistry (Scheme 1, method I), the stability of
S-geranyl-2-thiouridine was first tested under typical con-
ditions for RNA synthesis: geS2U was incubated in a 3%
trichloroacetic acid (TCA) in DCM, a 0.02 M iodine so-
lution, a mixture of Cap A and Cap B or in 0.25 M
protected with a mixture of Et3N·3HF in NMP. After pu-
rification (Figure 2A), 19 ODs of chromatographically pure
geS2U–RNA were obtained (ca. 55%). The molecular mass
(
MW 5483) of geS was confirmed by MALDI-TOF mass
spectrometry (m/z 5481) (Supplementary Figure S1).
Synthesis of geS via post-synthetic geranylation of CPG-
linked S2U-RNA (method II). An alternative route to geS
RNA involved the geranylation of 2-thiouridine in an al-
ready assembled oligomer still linked to the solid support
5
-(benzylmercapto)-1H-tetrazole (BMT) in ACN. Subse-
quent TLC analysis indicated that geS2U is sufficiently sta-
ble under these conditions. The stability of geS2U was fur-
ther analyzed under conditions commonly used for the re-
moval of base-labile protecting groups and for the hydrol-
ysis of succinyl linker. TLC analysis indicated that an aq.
ammonia/ethanol (3:1, v/v) solution is not suitable, as the
S-geranyl moiety is lost within 3 h of incubation at room
temperature. However, an alternative treatment with anhy-
drous 8 M NH3/EtOH for 8 h at room temperature did not
result in any visible degradation products. Furthermore, the
S-geranyl group remained intact after geS2U was treated
(
Scheme 1, method II). S-geranylation was previously re-
ported at the nucleoside level (11,14). The applied reaction
conditions (geranyl bromide, DIEA, methanol, 15 min.,
rt) seemed to be safe for canonical nucleosides, and we
performed a similar reaction at the oligomer level. The
S2U unit was incorporated into an RNA chain (E. coli
Lys
ASL UUU) using an appropriately protected S2U amidite
(
23) (2, Scheme 1). To avoid the loss of sulfur, a 0.25 M
tert-butyl hydroperoxide solution in toluene was used in
the oxidation steps (24). After some workup (see Materials
and Methods), the support-bound RNA was dried in vacuo
and S-alkylated with a mixture of geranyl bromide (>200
equivalents), TEA and EtOH (3 h, rt) (25). Further workup
and chromatographic purification furnished 13 ODs of geS
RNA (ca. 40% yield). IE-HPLC analysis of the crude prod-
uct (Figure 2B) indicated almost quantitative conversion of
S2U-RNA (Rt = 30.25 min.) to geS2U–RNA (Rt = 31.09
min.; see co-injection profile in Supplementary Figure S10).
The structure of geS2U–RNA and the selectivity of ger-
anylation were confirmed by MALDI-TOF mass spectrom-
etry (m/z 5481) as well as enzymatic hydrolysis followed
by analysis of nucleoside composition (Supplementary Fig-
ure S2A). RP-HPLC analysis revealed the presence of four
canonical nucleosides and the reference geS2U (at Rt = 54
min) (Supplementary Figure S2B).
.
with Et3N 3HF/NMP or 1 M TBAF/NMP (desilylating
conditions) for 24 h at room temperature.
Synthesis of S-geranyl-2-thiouridine phosphoramidite 1. S-
ꢁ
geranylated 5 -O-DMTr-2-thiouridine (5), obtained accord-
ing to a previously described procedure (14) and as shown
ꢁ
in Scheme 2, was further protected at the 2 -hydroxyl
group with tert-butyldimethylsilyl chloride (TBDMS-Cl)
under standard conditions (20,21), producing an equimo-
ꢁ
ꢁ
lar mixture of 2 - and 3 -O-TBDMS regioisomers. These
regioisomers were separated by column chromatography
ꢁ
and isomerization of the 3 -O-TBDMS regioisomer (in a
triethylamine/methanol solution), raised the overall yield
of 6 to 55 %. The structure of 6 was confirmed by H and C
NMR (Supplementary Figures S5 and S6) and by HRMS
1
13
(
(
FAB) mass spectrometry. A 2D COSY NMR experiment
ꢁ
Supplementary Figure S7) confirmed the identity of the 2 -
ꢁ
ꢁ
Synthesis of geS via post-synthetic geranylation of fully
deprotected S2U-RNA (method III). In this approach,
the synthesis of geS2U–RNA was based on the post-
synthetic geranylation of fully deprotected S2U–RNA
isomer via correlation of its H3 with 3 -OH protons.
Phosphitylation
anhydrous THF
of
6
was
performed
2-cyanoethyl
in
N,N-
using
diisopropylaminochlorophosphoramidite in the presence
of DMAP and DIEA (21,22). The product 1 was isolated
by silica gel flash column chromatography in 66% yield
(
Scheme 1, method III). The starting RNA, which con-
tained 2-thiouridine (17-mer E. coli ASL, S), was obtained
as described above. After final desalting, the product was
lyophilized, treated with an emulsion of geranyl bromide
and TEA in an ethanol/water solution, and then vigorously
shaken for 3 h at room temperature. After a pass through a
NAP-25 column, the crude geS2U–RNA product (geS) was
purified by IE-HPLC according to the procedure described
for methods I and II (Figure 2C). The identity of geS RNA
(obtained in ca. 67% yield) was confirmed by MALDI-TOF
1
31
and characterized by H and P NMR (Supplementary
Figures S8 and S9) as well as HRMS FAB.
Synthesis of geS via direct incorporation of geS2U unit
into the RNA chain (method I). The phosphoramidite
monomer 1 was used for the synthesis of geS RNA. Because
of the monomer’s modest solubility in ACN, a THF:ACN
mixture was used to prepare a monomer solution suitable

10992 Nucleic Acids Research, 2016, Vol. 44, No. 22
Scheme 1. A general scheme for geS2U–RNA synthesis. (I) direct incorporation of the geS2U unit using phosphoramidite 1; (II) post-synthetic chemical
geranylation of CPG-linked S2U–RNA with subsequent deprotection/cleavage from the solid support; (III) post-synthetic chemical geranylation of fully
deprotected S2U–RNA; (IV) enzymatic geranylation of fully deprotected S2U–RNA with the recombinant SelU enzyme.
Scheme 2. Synthetic pathway of S-geranyl-2-thiouridine phosphoramidite 1.
MS (m/z 5483, MW 5483, Supplementary Figure S3) and
by comparison with a genuine sample obtained via chemi-
cal synthesis (method I). The RP-HPLC chromatogram of
a mixture of S2U–RNA (Supplementary Figure S11A) with
its geranylated product (geS2U–RNA, Supplementary Fig-
ure S11B) shown in Figure 2D clearly confirms that the
presence of a geranyl residue increases the hydrophobicity
of geS over that of S.
Analysis of SelU catalytic activity
Preparation of recombinant SelU. SelU, a tRNA 2-
selenouridine synthase from E. coli, was previously de-
scribed as an enzyme that catalyzes two different reactions,

Nucleic Acids Research, 2016, Vol. 44, No. 22 10993
Figure 2. IE-HPLC analysis of the crude geS2U-RNA (geS) synthesized (A) via direct incorporation of geS2U unit with phosphoramidite 1 (method I,
anion-exchange IE-HPLC analysis on a Source 15Q 4.6/100PE® as described in the Materials and Methods); (B) via the post-synthetic geranylation
of support-linked S2U-RNA (method II, IE-HPLC as described above); (C) via post-synthetic geranylation of fully deprotected S2U-RNA (S) (method
III, anion-exchange IE-HPLC analysis on a Source 15Q 4.6/100PE® according to the methodology described in the Materials and Methods). The insets
ꢁ
ꢁ
indicate IE-HPLC analysis of purified oligoribonucleotides; (D) RP-HPLC co-injection of pure 5 -GUUGACUS2UUUAAUCAAC-3 (Rt = 11.0 min.)
ꢁ
ꢁ
®
and 5 -GUUGACUgeS2UUUAAUCAAC-3 (Rt = 17.3 min.) using a C18 column (Ascentis , 4.6 × 250 mm, 5 ␮m). The mobile phase composition
was as follows: 100% A (0.1 M ammonium acetate) for 5 min.; linear gradient of 100% buffer A to 100% buffer B (40% acetonitrile in 0.1 M ammonium
acetate) for 20 min.; maintain at 100% B for 5 min.
selenation (26) and geranylation of bacterial 5-(c)mnm-2-
thiouridine-containing tRNAs (11). We isolated the selU
gene (1095 bp) from total bacterial RNA and cloned it into
a pET28c expression vector between the sequence comple-
mentary to the T7 promoter and the region encoding a His6
affinity tag. The sequence of the insert was confirmed via
sequencing. The recombinant protein SelU-His6 (MW of
ca. 42 kDa) was overexpressed in BL21Star(DE3) and ac-
counted for ∼1% of the total bacterial protein pool (Supple-
mentary Figure S12). The protein was purified up to 90%
purity using HisPur Cobalt Resin according to the proce-
dure described in the Materials and Methods. An improved
preparation procedure allowed ∼1.5 mg of protein to be ob-
tained from 1 l of bacterial culture.
buffer (pH 8.0) with increasing amount of MgSO4. After 24
h of incubation at 25ºC, reaction mixtures were analyzed by
h
RP-HPLC (Figure 3). The geS2U–RNA product (geS ) ap-
peared as a single peak at Rt = 39.1 min. (the S2U–RNA
substrate peak was at Rt = 29.4 min). Samples collected
during RP-HPLC were analyzed by MALDI-TOF mass
spectrometry (Supplementary Figure S4), and the structure
h
of geS was confirmed (m/z 5477, MW 5483).
Enzymatic geranylations with thermally denaturated
2+
SelU or geranylations performed in Mg -free buffer were
unsuccessful (Figure 3A and B, respectively). Reactions car-
2+
ried out in buffers containing 10 or 100 mM Mg yielded
h
the desired geS product in 9 or 11 % yield (Figure 3C and
h
D), respectively. The geS oligomer was hydrolyzed with nu-
clease nP1 and calf intestine alkaline phosphatase. Nucleo-
side composition analysis by RP-HPLC then confirmed the
presence of the geS2U nucleoside at Rt = 49.4 min (Sup-
plementary Figure S13). The identity of the geS2U nucleo-
side was further confirmed via co-injection with an original
geS2U sample and via comparison of UV spectra (Figure
S14).
Enzymatic geranylation of S2U–RNA (method IV). Ap-
Lys3
proximately 40 ␮g (1 OD) of 17-mer oligo-RNA (ASL
)
ꢁ
ꢁ
with the sequence 5 -UCAGACUS2UUUAAUCUGA-3
Supplementary Figure S4A, m/z 5342, MW 5349) and con-
(
taining a single S2U substitution at the site corresponding
to the wobble position of the intact tRNA was incubated
with the SelU–His6 enzyme (10 ␮g) in the presence of a
5
-fold molar excess of the ammonium salt of geranyl py-
rophosphate. A set of reactions was performed in Tris-HCl

10994 Nucleic Acids Research, 2016, Vol. 44, No. 22
ꢁ
ꢁ
Lys3
h
Figure 3. RP-HPLC analysis of the S2U-RNA (5 -UCAGACUS2UUUAAUCUGA-3 , ASL of human tRNA
, S ) geranylation with (A) denatured
2+
2+
2+
SelU enzyme in the reaction mixture; (B) active SelU enzyme but without Mg ; (C) 10 mM Mg and active SelU enzyme; (D) 100 mM Mg and active
SelU enzyme. Rt of S = 29.4 min., Rt of geS = 39.1 min.
h
h
Thermodynamic stability of RNA/RNA and RNA/DNA du-
upon addition of silver ions (27), which mediate formation
of an additional bond in the geS/A duplex between the N3
in geS2U and the N1 in A (Figure 6). In this case, the Tm
of the geS/A duplex increased by 3.0 C, reaching the Tm
value of the geS/G duplex (Table 1).
plexes containing geS2U–RNA strands
To assess the thermodynamic stability of geS2U–
RNA/RNA and geS2U–RNA/DNA duplexes, UV-
monitored melting experiments were performed for a series
of 12 duplexes (Table 1). The U, S and geS RNA strands
◦
(
E. coli ASL with U, S2U or geS2U in the modification site,
Circular dichroism analysis of U, S and geS single stranded
RNAs and their duplexes
respectively) were annealed with complementary RNA or
DNA strands containing either A/dA or G/dG opposite
the modification site. Melting profiles are shown in Figure
The structures of U, S and geS single-stranded RNAs as
well as their duplexes with A and G matrices were analyzed
by CD spectroscopy. The measured spectra (Supplementary
Figure S16) indicate that both single stranded oligomers
◦
4
, and calculated Tm values as well as ꢀG values for ds→ss
dissociation (in kcal/mol) are given in Table 1. The full set
◦
◦
◦
of thermodynamic data (ꢀH , ꢀS and ꢀG ) is provided
in Supplementary Table S1. These results demonstrate that
the geranyl group in geS/A greatly decreases the stability
of the duplex compared to the U/A and S/A duplexes
containing U-A and S2U-A Watson–Crick base pairs):
the free Gibbs’ energy (ꢀG ) increased by approximately 5
(Supplementary Figure S16A) and their RNA/RNA du-
plexes (Supplementary Figure S16B and C) adopted typi-
cal A-type helical structures with a positive Cotton effect
maxima at ␭ ≈ 265 nm, a negative band at ␭ ≈ 210 nm and
a crossover point at 250 nm for ss RNA and ∼240 nm for
dsRNA. The S-geranyl group in the geS2U–RNA did not
disturb the overall A-type helical structure of the RNA. As
expected, we observed a shift of the crossover point from
(
◦
◦
kcal/mol, while Tm is reduced by approximately 9 C and
3 C compared to the U/A and S/A duplexes, respectively.
◦
1
The stability of the U/G and S/G duplexes (with U-G and
S2U-G wobble base pairs) was not changed significantly
by geranylation. The highest Tm value was observed for
the U/G duplex, while Tm values for S/G and geS/G were
2
40 to 260 nm in the case of RNA/DNA duplexes (Supple-
mentary Figure S16D and E), suggesting that the structure
of these duplexes is distorted from the A-RNA type. How-
ever, these duplexes remain more similar to the RNA rather
than DNA homoduplexes.
◦
◦
slightly decreased (by 1.5 and 3.5 C, respectively). ꢀG
for the S/G and geS/G duplexes were increased by 1.0
and 1.7 kcal/mol, respectively. Surprisingly, the geS/G
duplex was slightly more stable than the geS/A duplex,
◦
◦
with ꢀTm = 3.2 C, and ꢀꢀG = −1.5 kcal/mol. This
difference disappeared (see Supplementary Figure S15)

Nucleic Acids Research, 2016, Vol. 44, No. 22 10995
Figure 4. UV melting profiles for four sets of duplexes, (A) U/A, S/A and geS/A; (B) U/G, S/G and geS/G; (C) U/dA, S/dA and geS/dA; (D) U/dG,
S/dG and geS/dG.
Table 2. Analysis of the lipophilic properties of S-geranyl-2-thiouridine-
containing RNA (geS) in comparison to S2U-RNA (S) and unmodified
RNA (U) oligonucleotides
Thioalkyl groups are known as good leaving groups, so
we optimized our synthetic protocols to retain geS2U in the
assembled RNA chain. The geS2U unit was found to be sta-
ble under the conditions routinely used for automated solid
phase RNA synthesis. The desilylating conditions used also
left the S-geranyl group intact. We paid special attention to
the alkaline cleavage of the oligonucleotide from the solid
support and the removal of base-labile protecting groups
RNA oligonucleotide
Log P
U
− 1.74 ± 0.16
− 1.64 ± 0.13
− 1.47 ± 0.09
S
geS
(
from nucleobases and from phosphate moieties). Routine
treatment with aqueous ammonia/EtOH (3:1, v/v, 3 h incu-
Determination of the lipophilic properties of S-geranyl–RNA
oligonucleotides
bation, rt) was too harsh; fortunately, anhydrous conditions
(
8 M NH3/EtOH, 8 h, rt) did not cause any apparent degra-
The influence of a single geS2U modification on the hy-
drophobic properties of a 17-mer RNA oligonucleotide was
assessed by measuring the solubility of the oligonucleotide
in a water/1-octanol biphasic system and comparing this
measured solubility with the lipophilicities of reference U
and S RNA strands. The log P-values determined for U-,
S2U- and geS2U-containing oligo RNAs are given in Table
dation of geS2U. Furthermore, the geS oligonucleotide was
obtained with good yield (approximately 55% following iso-
lation by anion-exchange HPLC). In the second and third
methods (Scheme 1), we performed selective geranylation of
the sulfur atom in support-linked or released/deprotected
RNA oligomers containing 2-thiouridine. In method II,
detritylated CPG-linked S2U-RNA was alkylated with a
>200-fold molar excess (25) of geranyl bromide following
the removal of 2-cyanoethyl groups. Subsequent work-up
followed by IE-HPLC purification (Figure 2B) produced
the geS product with a yield (∼40%) similar to method I.
In method III, we successfully (∼67% yield) performed the
site-selective geranylation of fully deprotected S2U–RNA
using conditions similar to those used in previous approach.
The structure of geS and the site-selectivity of the post-
synthetic geranylation were confirmed by MALDI-TOF
mass spectrometry as well as by RNA enzymatic digestion
and nucleoside composition analysis as described in the RE-
SULTS section. It should be emphasized that, contrary to
reports in the literature (15), the S-geranylated oligomers
eluted later than the parent oligomers bearing the S2U unit
in our RP-HPLC analyses. This behavior should be ex-
pected for compounds decorated with a highly lipophilic
2
. As expected, a single geS2U modification increased the
lipophilicity of the entire RNA oligonucleotide by ꢀLog P
0.27.
=
DISCUSSION
To determine the properties of RNA chains bearing
S-geranylated units we synthesized two 17-nt long S-
geranylated RNAs, i.e. ASL RNAs with sequences homol-
Lys
Lys3
ogous to E. coli tRNA
(
(geS) or to human tRNA
h
geS ) and containing the geS2U modification in the site
corresponding to the wobble position. Three of the methods
used to prepare geS2U–RNA oligomers (methods I, II and
III) were purely chemical, while the fourth method involved
enzyme-catalyzed geranylation of S2U–RNA (Scheme 1).

10996 Nucleic Acids Research, 2016, Vol. 44, No. 22
ꢁ
geranyl substituent. The reported unexpected higher mo-
bility (shorter retention time in RP-HPLC) of the gerany-
lated product compared to its native precursor (T instead
of geS2T) was explained by the Authors as ‘a special struc-
tural alignment in the oligonucleotide’ (15). Our observa-
tions are in agreement with recently obtained log P data de-
termined at the level of R5geS2U nucleosides (R = H, mnm
or cmnm), which indicate a higher lipophilicity of these
compounds compared to their parent R5S2Us (16). In the
present studies, we measured the partition coefficients of the
U, S and geS oligonucleotides in a water/1-octanol biphasic
system. As expected, we found that a single geranyl modi-
fication increases the hydrophobicity of the geS2U–RNA
strand in comparison to the reference U (ꢀLog P = 0.27)
and S (ꢀLog P = 0.10) RNA strands (Table 2). Although it
has been suggested that the presence of a hydrophobic ger-
anyl residue in the tRNAs affects their intracellular local-
ization and transport through the cell membranes (11,15),
recent studies have shown a lack of geranylated tRNA en-
richment in outer or inner cellular membranes (9).
Several years ago, enzymatic S-geranylation of 2-
thiouridine in transfer RNAs was observed (11). In-
terestingly, this activity was eventually assigned to a
previously characterized enzyme, which is involved in
selenophosphate-dependent selenation of the same 2-
thiouridine–tRNA substrate (26). In E. coli, this enzyme
was named tRNA selenouridine synthase (SelU, also named
MnmH, SufY or YbbB). In earlier studies, we chemi-
cally transformed S2U to Se2U via S-geranyl-2-thiouridine
and suggested a possible linear mechanism for the SelU-
catalyzed reaction, in which geS2U exists as an intermedi-
ate in the cellular transformation of S2U to Se2U (Figure
the geS2U wobble units in recognizing the 3 -ending purines
in synonymous mRNA codons (for Lys, Glu and Gln). We
were able to demonstrate via UV-melting measurements of
various RNA/RNA and RNA/DNA duplexes that the ger-
anyl modification decreases the thermodynamic stability of
the geS/A duplexes in comparison with the S/A and U/A
duplexes, which contain S2U-A and U-A Watson–Crick
base pairs. In contrast, the stability of the S/G and U/G du-
plexes containing S2U-G and U-G wobble base pairs does
not differ significantly upon S2U geranylation. Moreover,
the geS/G duplex is more stable than the geS/A duplex.
The higher thermal stability of a model 11-bp DNA du-
plex containing a geS2T-dG base pair compared to a duplex
containing a geS2T-dA base pair was demonstrated previ-
ously (15). All these data indicate that geranylation changes
the reading preferences from A to G. This phenomenon has
been attributed to changes in the hydrogen bonding proper-
ties of the S-geranylated 2-thiouridine via conversion of the
N3H donor into an N3 acceptor site (Figure 6). A similar
acceptor pattern could be observed in 4-pyrimidinone ribo-
side (H2U), the product of S2U oxidative desulfuration (8).
We have already shown that duplexes made of H2U–RNA
with RNA or DNA strands are more stable when H2U is
base-paired with G rather than A (6–8,29). This H2U-like
2-fold bond system is also present in geS2U and in prestruc-
tured (c)mnm5S2U nucleosides (30, Sochacka et al. submit-
ted). These nucleosides all hybridize preferably to guano-
sine. Therefore, this new arrangement between the modi-
fied wobble nucleosides and guanosine offers precise recog-
ꢁ
nition of synonymous 3 -G-ending codons.
The stability of the geS/A duplex is low because only
one hydrogen bond is present between the O4 acceptor and
HN6 donor centers. In addition, repulsive interactions are
present between the N3 of geS2U and N1 of A. Our earlier
modeling studies showed that the H2U-A base pair in RNA
duplexes is bound by this single hydrogen bond (8). Adding
silver ions, which mediate the formation of bonds between
acceptor centers with lone ion pairs (31), resulted in the
formation of an additional bond in the geS2U-A base pair
5) (14). Such a possibility has been recently considered in
advanced studies on tRNA selenouridine synthase (9).
The SelU enzyme exhibits intrinsic low geranylation ac-
tivity (∼10%) (10). However, some MnmH proteins with
mutations in the rhodanese domain exhibit improved tRNA
geranylation efficiences (9). We measured the geranylation
potential of a recombinant SelU-His6 enzyme on a human
Lys3
h
◦
ASL
S2U-RNA model (S ). The geranylation reaction,
(Figure 6) and an increase in Tm (3.0 C, Table 1). This result
carried out in the presence of an excess of SelU–His6 and
geranyl pyrophosphate, was not successful unless magne-
sium ions were added to the reaction mixture. Despite our
efforts at improvement, the yield of enzymatically gerany-
confirms the presence of two acceptor sites at the geS2U-A
base pair.
Our circular dichroism analysis demonstrated that the S-
geranylated unit in the RNA chain does not alter the struc-
ture of the single stranded geS chain and does not affect the
overall conformation of the RNA/RNA duplexes, which
preferentially adopt typical A-type helical structures. As ex-
pected, the structures of RNA/DNA duplexes are distorted
from that of the A-RNA type; however, the structures of the
duplexes remain more similar to that of the RNA homodu-
plex rather than the DNA homoduplex.
In conclusion, we describe four different methods of
preparing S-geranylated RNA based on chemical and en-
zymatic site-specific geranylation of the sulfur atom in 2-
thiouridine. The recombinant SelU-His6 protein accepts a
simplified tRNA model consisting of the S2U-ASL domain
but only in the presence of magnesium ions at millimo-
lar concentrations. We demonstrate that the S-geranyl-2-
thiouridine-containing model exhibits enhanced hydropho-
bicity compared to its parent S2U–RNA and non-modified
2+
lated S2U–RNA was rather low (9% with 10 mM Mg and
2+
1
1% with 100 mM Mg ) (Figure 3). We assume that our
S2U–RNA used for enzymatic geranylation only weakly
mimics natural tRNAs, as it is limited to the anticodon-
stem-loop domain. Moreover, this model does not have a 5-
(
c)mnm side chain at 2-thiouridine, an important structural
element for the geranylating activity of tRNA selenouridine
synthase in vivo (9). The enzymatic activity of SelU may be
lower because the enzyme contains a bound tRNA popula-
tion in its native form, and a part of the bound tRNA popu-
lation is S-geranylated. Therefore, the exchange of naturally
bound tRNA (to which the enzyme exhibits high affinity)
with a poor S2U–RNA substrate is disfavored unless mag-
nesium ions are added; magnesium ions likely facilitate the
formation of better accepted and structured ASL molecule
(
28).
The more fundamental question relates to the function of

Nucleic Acids Research, 2016, Vol. 44, No. 22 10997
Figure 5. Proposed linear (blue arrow) and bidirectional (red arrows) pathways of SelU activity on the S2U–tRNA substrate.
Figure 6. Base pairing modes of geS/A, geS/G and geS/A with Ag+.
RNA. Moreover, the S-geranyl-2-thiouridine-RNA dis-
plays improved affinity for RNA matrices with a G oppo-
site the modification site. The geS/A duplex is thermody-
namically less stable. These results further support the al-
ready proven biological property of S-geranylated tRNAs,
Conflict of interest statement. None declared.
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