Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine

Article abstract of DOI:10.1093/nar/gkr673

The synthesis of a caged RNA phosphoramidite building block containing the oxidatively damaged base 5-hydroxycytidine (5-HOrC) has been accomplished. To determine the effect of this highly mutagenic lesion on complementary base recognition and coding properties, this building block was incorporated into a 12-mer oligoribonucleotide for T_m and CD measurements and a 31-mer template strand for primer extension experiments with HIV-, AMV-and MMLV-reverse transcriptase (RT). In UV-melting experiments, we find an unusual biphasic transition with two distinct T_m's when 5-HOrC is paired against a DNA or RNA complement with the base guanine in opposing position. The higher T _m closely matches that of a C-G base pair while the lower is close to that of a C-A mismatch. In single nucleotide extension reactions, we find substantial misincorporation of dAMP and to a lesser extent dTMP, with dAMP almost equaling that of the parent dGMP in the case of HIV-RT. A working hypothesis for the biphasic melting transition does not invoke tautomeric variability of 5-HOrC but rather local structural perturbations of the base pair at low temperature induced by interactions of the 5-HO group with the phosphate backbone. The properties of this RNA damage is discussed in the context of its putative biological function.

Full text of DOI:10.1093/nar/gkr673

9422–9432 Nucleic Acids Research, 2011, Vol. 39, No. 21
doi:10.1093/nar/gkr673
Published online 18 August 2011
Synthesis, base pairing properties and trans-lesion
synthesis by reverse transcriptases of
oligoribonucleotides containing the
oxidatively damaged base 5-hydroxycytidine
Pascal A. Ku¨ pfer and Christian J. Leumann*
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland
Received June 18, 2011; Revised and Accepted July 29, 2011
(5-HOdU) and 5-hydroxy-dC (5-HOdC) (Figure 1).
Some of them can be highly mutagenic during DNA rep-
lication in vivo (1–3). For example, 8-oxo-dG is known to
lead to a substantial level of G-T transversions (4). On the
other hand 5-HOdU, originating from oxidative deamin-
ation of dC via the intermediates 5,6-dihydroxy-5,
6-dihydro-2’-deoxycytidine (dCg) and 5,6-dihydroxy-
5,6-dihydro-2’-deoxyuridine (dUg), causes high levels of
C!T transition mutations (5). While there is some con-
troversy on the level of mutagenicity of the intermediates
along the oxidation pathway (5–7), it has been shown in
Escherichia coli that 5-HOdC itself also causes C!T tran-
sitions, however, at a significantly lower frequency (5).
The mutagenicity has been associated with tautomeric
variability of the oxidized base (7–9).
Nucleobase damage by ROS, however, is not restricted
to DNA but also occurs in RNA. It is conceivable that the
base lesions in RNA are the same as that in DNA. Indeed,
8-oxo-rG (10), as well as 8-oxo-rA, 5-hydroxy-U
(5-HOrU) and 5-hydroxy rC (5-HOrC) (11), have been
isolated from RNA and identified in the past. RNA is
even more prone to base oxidation than DNA for the
following reasons: (i) the relative abundance of RNA in
a cell is higher compared to DNA; (ii) RNA is generally
less protected by proteins; and (iii) RNA is not only
localized in the nucleus but also in the cytosol where the
exposure to ROS, specifically near the mitochondria, is
significantly higher (12). While nature has developed a
highly efficient DNA repair machinery to protect the
genome, the mechanism of which has been intensively
studied in the past and is now fairly well understood,
the impact on cellular function of base damaged RNA
has remained poorly investigated in the past, presumably
due to the assumption of its transient nature and its high
copy numbers. Moreover, its cellular metabolism (clear-
ance or repair) is virtually unknown.
ABSTRACT
The synthesis of a caged RNA phosphoramidite
building block containing the oxidatively damaged
base 5-hydroxycytidine (5-HOrC) has been accom-
plished. To determine the effect of this highly muta-
genic lesion on complementary base recognition
and coding properties, this building block was
incorporated into a 12-mer oligoribonucleotide for
Tm and CD measurements and a 31-mer template
strand for primer extension experiments with HIV-,
AMV- and MMLV-reverse transcriptase (RT). In
UV-melting experiments, we find an unusual
biphasic transition with two distinct Tm’s when
5-HOrC is paired against a DNA or RNA complement
with the base guanine in opposing position. The
higher Tm closely matches that of a C-G base pair
while the lower is close to that of a C-A mismatch. In
single nucleotide extension reactions, we find sub-
stantial misincorporation of dAMP and to a lesser
extent dTMP, with dAMP almost equaling that of
the parent dGMP in the case of HIV-RT. A working
hypothesis for the biphasic melting transition does
not invoke tautomeric variability of 5-HOrC but
rather local structural perturbations of the base
pair at low temperature induced by interactions of
the 5-HO group with the phosphate backbone.
The properties of this RNA damage is discussed
in the context of its putative biological function.
INTRODUCTION
Oxidative damage to DNA bases by reactive oxygen
species (ROS) such as hydrogen peroxide, superoxide
and hydroxyl radicals are recognized as a major threat
to the integrity of the genome. The most abundant base
lesions known are 8-oxo-dA, 8-oxo-dG, 5-hydroxy-dU
Oxidatively damaged mRNA is known to cause largely
diminished translation efficiency and abnormal protein
synthesis, both in vitro and in vivo (13). Given the high
*To whom correspondence should be addressed. Tel: +41 31 631 4355; Fax: +41 31 631 3422; Email: leumann@ioc.unibe.ch
ß The Author(s) 2011. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acids Research, 2011, Vol. 39, No. 21 9423
Figure 1. Chemical structures of the main base lesion products arising from reactive oxygen species in DNA and RNA.
oxygen turnover in the brain, the RNA in neural cells
is particularly exposed to ROS. It is therefore no
surprise that damaged RNA is related to a series of
neurodegenerative diseases such as Alzheimer’s and
Parkinson’s disease (14). Particularly in the light of its
various functions which are not restricted to the transfer
of genetic information from DNA to proteins only
(tRNA, rRNA and mRNA), but also include the control
of gene expression (non-coding RNAs, miRNAs), a more
thorough investigation of the function of base-damaged
RNA would be highly welcome.
To understand the properties and the interactions
of such damaged bases in more detail, 8-oxo-rA,
8-oxo-rG and 5-HOrU have recently been incorporated
into oligoribonucleotides by synthetic means and their
base pairing properties with complementary RNA as
well as their coding preferences in template–primer
extension reactions by reverse transcriptases (RTs) have
been studied (15–17). Despite its high mutagenicity the
base recognition and coding properties of the 5-HOrC
lesion has only scarcely been investigated in DNA in
detail (18), and even less in RNA. In early work, poly
5-hydroxycytidylic acid, prepared from 5-HOrCDP by
polynucleotide phosphorylase, was reported to show
little secondary structure in neutral and acidic solution
and no complex formation with poly(G), poly(I) and
poly(A) (19).
the base lesion product 5-HOrC, on its base recognition
properties as determined by UV-melting curves, on its
structural properties inferred from CD spectroscopy as
well as on its coding preferences in primer/template exten-
sion reactions with RTs of three different organisms.
MATERIALS AND METHODS
Oligonucleotide synthesis and purification
Unmodified DNA and RNA oligonucleotides were from
Microsynth (Balgach, Switzerland) and HPLC-purified
where necessary. A 12-mer oligoribonucleotide (50-AUG
CUXAGUCGA-30) and a 31-mer template oligoribo-
nucleotide (50-UAGUCUGCACXUGCACCAGUCGCU
CAGGGAU-30) were synthesized using building block 6
for X, the natural 20-TOM protected RNA phos-
phoramidites and polystyrene (GE Healthcare) or CPG
solid supports (GlenResearch). The syntheses were per-
formed on a 1.3 mmole scale on a Pharmacia Gene
Assembler Plus DNA synthesizer following standard phos-
phoramidite protocols with 5-(ethylthio)-1H-tetrazole
(0.25 M in CH3CN) as the activator and coupling times
of 12 and 6 min for building block 6 and unmodified RNA
phosphoramidites, respectively. Oligoribonucleotides were
deprotected using 33% NH3/EtOH (3:1) for 16 h at
55ꢀC. Solid supports were then filtered off and washed
with water (CPG solid support) or EtOH/CH3CN/H2O
3:1:1 (polystyrene solid support). The solutions were
In an effort to elucidate the biological impact on base
recognition, RT and translation of base-damaged RNA,
we recently prepared abasic RNA and investigated its
chemical stability as well as its trans-lesion synthesis by
RTs in a comparative study with DNA (20–22). Here, we
report on the synthesis of oligoribonucleotides containing
evaporated to dryness at room temperature in
a
Speedvac evaporator. The resulting pellets were further
dried by addition and evaporation of dry ethanol. The
pellets were then dissolved in anhydrous DMSO (100 ml)

9424 Nucleic Acids Research, 2011, Vol. 39, No. 21
at 65ꢀC and neat triethylamine trihydrofluoride (125 ml)
was added. After shaking at 65ꢀC for 90 min (or at 25ꢀC
for 24 h for modified oligoribonucleotides), an aqueous
solution of NaOAc (3 M, 25 ml) and n-butanol (1 ml)
were added and the mixture was chilled on dry ice for
45 min. After centrifugation for 20 min in an Eppendorf
centrifuge the liquid was discarded and the pellet washed
twice with chilled ethanol (80%, 0.75 ml) and dried under
high vacuum at room temperature, yielding the oligo-
nucleotides, in which the nitrobenzyl protecting group
on the 5-HOrC unit was still in place (X = 5-nbnOrC).
The 31-mer oligoribonucleotide was purified on a prepara-
tive 20% denaturing polyacrylamide gel, the product
band electroeluted with an Elutrap electroelution system
(Schleicher & Schuell) and desalted over NAP-10 columns
(GE Healthcare). The 12-mer was HPLC purified on a
RT assays
RT assays were performed with HIV-1 RT (Worthington
Biochemical Corp), AMV RT (Promega Corp.) and
MMLV RT (USB Corp./Affymetrix Inc.,) in the following
buGers: HIV-1 RT and AMV RT: 50 mM Tris (pH 8.3),
50 mM NaCl, 8 mM MgCl2, 1 mM DTT; M-MLV RT:
50 mM Tris (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM
DTT. Final reaction mixtures contained RNA template
(100 nM), DNA primer (50 nM) and dNTP (20 mM).
After addition of enzyme (0.5–40 U), the mixtures were
incubated at 37ꢀC for 1 h. The reactions were quenched
with gel loading buGer (98% formamide, 0.05% xylene
cyanol (FF), 0.05% bromophenol blue), heated to 90ꢀC
for 5 min and applied to a 20% denaturing polyacrylamide
gel. Radioactivity was detected and quantiEed on a Storm
820 phosphorimager with ImageQuant software (GE
Healthcare).
DNAPac-200 column (4 ꢁ 250 mm, Dionex) with
a
gradient of solvent A (25 mM Tris, pH 8.0) and solvent
B (25 mM Tris, 1.25 M NaCl, pH 8.0), 0–50% B in 30 min
at room temperature, and finally desalted on a Sep-Pak
C18 column (Waters Corp.).
RESULTS
Synthesis of phosphoramidite 6
Deprotection of the 2-nitrobenzyl group was achieved
by irradiating aliquots of template (300 ml, 50 mM) or
12-mer (300 ml, 200 mM) in H2O for 1 h using a slide pro-
jector (25 W tungsten lamp) as described before (20).
Wavelengths below 300 nm were filtered off by a glass
filter set between the samples and the projector lamp.
The fully deprotected oligoribonucleotides (X =
5-HOrC) were then purified by IE-HPLC as described
above. Oligonucleotide concentrations were determined
using a NanoDrop ND-100 UV/Vis spectrophotometer
(NanoDrop Technologies, Inc.). Stock solutions were
made from DEPC treated H2O.
While the synthesis of a phosphoramidite of 5-HOdC and
its incorporation into DNA was already described in the
literature (23,24), the phosphoramidite of the RNA
building block was not known and had first to be accom-
plished. Starting from cytidine (1), oxidation at position 5
of the base was achieved in analogy to earlier work with
Br2 and H2O (19) giving 5-HOrC (2) (Scheme 1). After
standard 50-O-tritylation, we first attempted to protect
the 5-OH group on the base by acylation in analogy to
the known DNA building block. While acetylation or
benzoylation was in principle successful, both ester func-
tions were rather labile leading to substantial hydrolysis
during chromatography. This was also the case for silyl
protecting groups such as tert butyldimethylsilyl
(TBDMS) or tert butyldiphenylsilyl (TBDPS). We there-
fore switched to the photochemically cleavable
2-nitrobenzyl group which was smoothly introduced
yielding nucleoside 3. This orthogonal protecting group
offers the possibility to study oligoribonucleotides in
which 5-O is still protected and therefore not ionizable.
The 2-nitrobenzyl ether was stable and no specific
measures for light protection had to be taken during syn-
thetic manipulations. The N4-amino group of the base
was subsequently protected as a formamidine giving nu-
cleoside 4 in excellent yield. The 20-hydroxy group of the
ribose was silylated with TBDMS-Cl in the presence of
pyridine and AgNO3, leading to a 2:1 mixture of 20-O-,
and 30-O-silylated compounds 5a and b that could be
separated by column chromatography and the structures
unambiguously assigned by 1H,1H-COSY-NMR spectros-
copy. Building block 6 for RNA synthesis was finally
achieved by standard phosphitylation of 5a. The experi-
mental details and the analytical data of compounds 2–6
are given in the Supplementary Data.
UV-melting curves
Solutions were prepared in standard saline buffer (10 mM
NaH2PO4, 150 mM NaCl, pH 7.0) with duplex concentra-
tions of 2 mM in 1:1 strand ratio. Thermal melting experi-
ments were carried out on a Varian Cary 100-Bio UV/VIS
spectrophotometer (Varian Inc.), equipped with a Peltier
element at 260 nm with a heating/cooling rate of 0.5ꢀC/
min Tm values were obtained from the maxima of the Erst
derivatives of the melting curves using WinUV software.
Labeling of oligonucleotides
The DNA primer was phosphorylated with [g-32P]ATP
(Hartmann Analytic) and T4 polynucleotide kinase
(Fermentas) as follows: DNA primer (30 pmol),
[g-32P]ATP (60 mCi) and T4 PNK (10 U) were incubated
in a total volume of 20 ml at 37ꢀC for 30 min in T4 PNK
buffer (50 mM Tris–HCl, pH 7.6, 10 mM MgCl2, 5 mM
DTT, 0.1 mM spermidine, 0.1 mM EDTA). T4 PNK was
inactivated by heating the sample to 90ꢀC for 2 min. The
labeled primer was then directly annealed to the RNA
template (60 pmol) in the reaction buffer of the respective
enzyme (see ‘RT assays’) by heating to 60ꢀC for 5 min and
slow cooling to room temperature.
Synthesis of oligoribonucleotides
Two sets of oligoribonucleotides containing either a 5-O
nitrobenzyl protected (5-nbnOrC) or a free 5-HOrC unit,

Nucleic Acids Research, 2011, Vol. 39, No. 21 9425
Scheme 1. (a) 1. Br2, H2O, 0ꢀC; 2. sym-collidine, 40ꢀC, 2 h; 64%; (b) 4,40-dimethoxytriphenyl chloride, AgNO3, pyridine, 3.5 h, rt; (c) 2-nitrobenzyl
bromide, K2CO3, NaI, acetone, 60ꢀC, 3 h, 64% over two steps; (d) N,N-dimethylformamide dimethyl acetal, DMF, 60ꢀC, 1 h, 99%; (e) tert-
butyldimethylsilyl chloride, AgNO3, pyridine, THF, 2 h, 50% for 5a; (f) 2-cyanoethyl diisopropylchlorophosphoramidite, N-ethyldiisopropylamine,
THF, rt, 16 h, 71%. DMT = 4,40-dimethoxytriphenyl, TBDMS = tert-butyldimethylsilyl.
a 12-mer for Tm and CD measurements and a 31-mer
template strand for primer extension reactions were
synthesized using building block 6. Coupling yields
as determined by trityl assay were >98% at 12 min
coupling time. Oligoribonucleotides were first base-, phos-
phate and 20-O deprotected yielding the oligoribonu-
cleotides with X = 5-nbnOrC that were purified, and
their composition verified by mass spectrometry
(Supplementary Table S1). The nitrobenzyl group was
then photolytically cleaved by a tungsten slide projector
lamp (l >300 nm) leading to the fully deprotected
oligoribonucleotides (X = 5-HOrC). This reaction is
clean and goes to completion within 1 h as can be seen
from Figure 2 which shows the HPLC traces of the
31-mer template before and after nitrobenzyl deprotection
as an example. Successful deprotection was again
controlled by mass spectrometry (Supplementary
Table S1). The half-live of nitrobenzyl deprotection was
determined with a 12-mer oligodeoxyribonucleotide of the
same sequence as above, and found to be 11.4 min under
the conditions applied (Supplementary Figure S1). Fully
deprotected oligoribonucleotides were again purified by
HPLC and used as such in the following experiments.
those of the unmodified duplexes (X = rC, Figure 3).
The corresponding Tm data are summarized in Table 1.
Melting of the 12-mer with and without nitrobenzyl pro-
tecting group in the absence of complementary strands
gave no sigmoidal transition, thus excluding self-structure
formation of the single strand (data not shown). From the
Tm data it becomes clear that the 5-nitrobenzyl protected
12-mer (X = 5-nbnOrC) shows strong destabilization
when paired against rA, rC or rU (Tm = 44.9–47.1ꢀC)
but only a slight destabilization compared to the unmodi-
fied duplex (X = rC) when paired against rG (ÁTm
5-nbnOrC versus rC = ꢂ3.9ꢀC). The same trend albeit
with generally lower Tm’s is also observed if the comple-
ment is DNA. Thus 5-nbnOrC behaves by and large as
cytosine in nucleobase recognition.
The situation is similar in the case of the fully
deprotected 12-mer (X = 5-HOrC) when paired against
rA, rC or rU, but significantly different when paired
against rG. In this case two transitions were observed in
which Tm1 (47.7ꢀC) closely matches that of a complemen-
tary rC-rA base mismatch whereas Tm2 (65.2ꢀC) is remin-
iscent of that of a unmodified duplex with a matched
rC-rG base pair. The equivalent behavior was found
again with DNA as complement. Also here, two transi-
tions were observed only with dG as the complementary
base. This biphasic melting behavior could be reproduced
with different samples over multiple heating and
UV-melting curves
Melting curves of the 12-mers were measured at 260 nm
with complementary RNA and DNA and compared to

9426 Nucleic Acids Research, 2011, Vol. 39, No. 21
Figure 2. HPLC traces of 31-mer (50-UAGUCUGCACXUGCACCAGUCGCUCAGGGAU-30) before (trace A, X = 5-nbnOrC, Rt = 29.3 min) and
after (trace B, X = 5-HOrC, Rt = 28.7 min) nitrobenzyl deprotection; trace C: coinjection of both.
cooling cycles with some variations on the relative
hyperchromicities of the two transitions (Supplementary
Figures S4–S5).
A CD-spectroscopic investigation was performed to de-
termine whether pairing of 5-HOrC against rA or rG is
associated with structural differences in comparison to the
unmodified duplexes with a rC-rA mismatch or a rC-rG
match (Figure 4). All CD spectra are very similar and
suggest classical A-duplex conformation. The only differ-
ence is a slightly decreased positive cotton effect at 263 nm
in both duplexes containing the 5-HOrC residue which is
most likely due to differences in the UV absorption
spectra between rC and 5-HOrC (see Supplementary
Data). Thus the CD spectra suggest no major structural
perturbations imposed by the 5-HOrC unit, irrespective of
whether the opposing base is A or G.
To exclude deamination and thus 5-HOrU formation
to occur under Tm-conditions we incubated in a control
experiment the free nucleoside 2 in Tm buffer for
24 h at 80ꢀC and compared the 1H and 13C-NMR
spectra with those of 5-HOrU from an authentic sample.
In the carbon NMR we could identify small signals arising
from 5-HOrU, however, at an abundance of <5%
(Supplementary Figures S2 and S3) suggesting that
5-HOrC to 5-HOrU conversion during Tm measurements
is negligible. We also measured melting curves with
duplexes containing an authentic 5-HOrU unit paired
against G or A (X = 5-HOrU, Table 1), the Tms of
which are consistently different from that of the 5-HOrC
containing duplexes. Based on these results we rule out
partial hydrolysis of 5-HOrC to be the cause for the
biphasic melting behavior.
To investigate whether the two transitions are
associated with different ionization states of the 5-OH
group we measured Tm’s with complementary rG and
dG at pH 5.5, 7.0 and 8.5 (Supplementary Figures S4
and S5). In all cases, we found two transitions. The
relative intensities of the hyperchromicities of these two
transitions between continuous rounds of heating and
cooling were lower at pH 5.5 as compared to pH 7.0 or
8.5. The two Tm’s were always of the same magnitude as
described in Table 1, irrespective of the pH. We thus
conclude that there are no fundamental pH-dependent
differences in this range, perhaps with the exception
that at pH 5.5 the relative differences between the
hyperchromicities of the two transitions between consecu-
tive cooling/heating cycles are smaller as compared to the
measurements at higher pH.
Primer template extension experiments
To elucidate the readout of the 5-HOrC lesion by RTs, we
performed standing start primer extension reactions
on the 31-mer template with a corresponding 50-32P-
radiolabeled DNA primer, dNTPs and three different
RTs that vary in their transcription fidelity (HIV-RT,
AMV-RT and MMLV-RT, Figure 5). Quantitative data
of dNTP incorporation opposite the lesion are given in the
Supplementary Table S2.
As expected all three RTs readily insert dGMP opposite
the 5-HOrC lesion with high efficiency, and all enzymes
proceed to full-length extension in the presence of all 4
dNTPs (Figure 5, top). But all three enzymes also insert
a dAMP residue to a substantial amount opposite the
lesion. In the case of HIV-RT, this proceeds about as ef-
ficiently as dGMP incorporation (63% for dAMP versus
64% for dGMP) while for the other two enzymes this
process is ꢃ2- to 3-fold less efficient. Interestingly,
HIV-RT inserts also dTMP with about half of the effi-
ciency of dGMP while AMV- and MMLV-RT

Nucleic Acids Research, 2011, Vol. 39, No. 21 9427
Figure 3. Tm curves for duplexes of 50-AUGCUXAGUCGA-30 with complementary RNA (left column) and complementary DNA (right column)
displaying all four natural nucleobases opposite X; top row: X = 5-nbnOrC; middle row: X = rC and bottom row: X = 5-HOrC; duplex conc.: 2 mM
in 10 mM NaH2PO4, 150 mM NaCl, pH 7.0.
Table 1. Tm data (ꢀC, 260 nm) of duplexes of 50-AUGCUXAGUCGA-30 with 50-UCGACUYAGCAU-30 (RNA) or 50-TCGACTYAGCAT-30
(DNA) as complement; duplex concentration: 2 mM in 10 mM NaH2PO4, 150 mM NaCl, pH 7.0
X
RNA complement (Y)
DNA complement (Y)
rG
rA
rC
rU
dG
dA
dC
dT
rC
65.7
61.8
65.2 47.7
54.6
54.2
47.8
47.1
47.4
59.2
58.8
45.6
44.8
44.0
–
46.0
44.9
44.6
–
49.9
43.6
48.6 25.9
32.0
32.5
28.4
31.3
27.2
42.3
42.6
26.7
28.1
25.4
–
27.6
28.3
25.4
–
5-nbnOrC
5-HOrC, Tm1 Tm2
rU
5-HOrUa
–
–
–
–
aSynthesized in analogy to known methods (15).

9428 Nucleic Acids Research, 2011, Vol. 39, No. 21
Figure 4. CD spectra of 12-mer duplexes: left: 50-AUGCUXAGUCGA-30 and 50-UCGACUAAGCAU-30 with X = rC or 5-HOrC; right:
50-AUGCUXAGUCGA-30 and 50-UCGACUGAGCAU-30 with X = rC or 5-HOrC. Duplex concentration: 2 mM (1:1 strand ratio) in 10 mM
NaH2PO4, 150 mM NaCl, pH 7.0; T = 25ꢀC.
incorporate only traces of dTMP (ꢃ10%) relative to
dGMP. The remaining pyrimidine nucleotide, dCMP, is
hardly incorporated by any of the three enzymes. Thus the
order of preferential incorporation is G ꢃ A>T>>C for
HIV-RT and G>A>T>>C for AMV-RT and
MMLV-RT.
To determine how an alkyl residue at 5-O in the base,
rendering it non-ionizable, influences the coding
properties we performed the same experiments on the
template with X = 5-nbnOrC (Figure 5, center). Again,
full-length extension of the primer in the presence of all
dNTPs occurs readily with all three enzymes. The order
and efficiency of incorporation of the single dNTPs
follows closely that observed for the 5-HOrC containing
template in the case of AMV- and MMLV-RT. With
HIV-RT, however, the extent of misincorporation of dA
is reduced compared to 5-HOrC.
The same experiment with a template containing a
natural rC unit as a control (Figure 5, bottom) also
reveals significant dAMP and dTMP misincorporation
with HIV-RT. However, the extent of misincorporation
of dA is significantly reduced compared to 5-HOrC
(49% for dAMP versus 77% for dGMP). Insertion of
dT proceeds for all bases investigated to about the same
extent. Again, MMLV- and AMV-RT display higher
fidelity with almost exclusive incorporation of dGMP
and only traces of dAMP and dTMP.
These experiments clearly show that there is substantial
miscoding propensity for dA and to a lesser extent also for
dT during cDNA synthesis opposite 5-HOrC by all three
RTs investigated. For HIV-RT, the ratio of incorporation
of dA versus dG opposite to 5-HOrC is approximately
0.98 whereas for rC it is 0.63. That of dT is about equal.
Interestingly, the presence of a substituent at O-5 of the
base, as in 5-nbnOrC, shows only slightly enhanced
misincorporation of dAMP compared to rC, suggesting
that either the ionization state or the proton itself in
5-HOrC influences the coding preferences in a subtle
way. Our results are also in agreement with earlier work
on primer template extension in DNA with the Klenow
fragment of DNA polymerase I, where in one sequence
context also increased misincorporation of dA opposite
5-HOdC was observed.
Our results reflect similar behavior to standing start
primer template extension reactions on a DNA template
by Klenow fragment (Kf) DNA polymerase, where also
substantial misincorporation of dAMP was observed
opposite 5-HOdC and to a much lesser extent opposite a
natural dC residue (18).
DISCUSSION
In this article, we describe for the first time the incorpor-
ation of the RNA lesion product 5-HOrC into oligoribo-
nucleotides by RNA phosphoramidite chemistry. The
synthetic problem that had to be solved was the choice
of an appropriate protecting group for the 5-OH group.
Standard acyl groups such as benzoyl used for the corres-
ponding 20-deoxynucleoside building block (23,24), or silyl
groups proved too labile in our hands. This is likely due
to the rather high acidity of this phenolic hydroxyl
group [pKa 7.37 in the case of the corresponding
20-deoxynucleoside (9)], which renders it a good leaving
group and the ester an activated ester. The choice of the
caged benzyl group used in this study thus fulfills two
purposes: (i) it confers chemical stability during building
block and oligoribonucleotide synthesis and (ii) it is
orthogonal in its deprotection conditions to the other
RNA protecting groups which allows also the isolation
of the 5-O-substituted oligonucleotides. This can be
of interest for further studies where protonation state
dependent tautomerism (7), or the redox chemistry of
this base in the context of oligoribonucleotides is of
interest (25).
Clearly the most striking result of this work is the
biphasic melting behavior of duplexes with
a
5-HOrC-rG or a 5-HOrC-dG base pair, giving rise to
two Tm’s where the higher one is close to that of a
matched rC-rG or rC-dG base pair, respectively, while
the lower one is close to that of a mismatched
5-HOrC-rA or 5-HOrC-dA pair, respectively. The
absence of this effect in duplexes where 5-HOrC is

Nucleic Acids Research, 2011, Vol. 39, No. 21 9429
Figure 5. Autoradiograms of standing start primer extension reactions for the reverse transcriptases indicated. Reactions were performed at 37ꢀC for
1 h. Enzyme concentrations: HIV-RT, 0.5 U; AMV-RT, 8 U; MMLV-RT, 40 U. Ref: primer without enzyme; A, T, G, C: reactions in presence of
the according dNTP; N: reactions in presence of all four dNTPs; Nat: unmodified RNA template (X = U) and all four dNTPs.

9430 Nucleic Acids Research, 2011, Vol. 39, No. 21
While the amino form emulates the hydrogen bonding
pattern of C the imino form emulates that of T(U).
Theoretical calculations in the gas phase favor the
amino over the keto tautomer in the neutral form (8).
UV resonance Raman spectroscopy of 5-HOdC shows
enhanced presence of the imino tautomer by ꢃ100-fold
compared to dC at physiological pH and temperature,
which results in a frequency of occurrence of this
tautomer of 0.3–0.7%. The tendency toward the imino
form slightly increases with temperature and pH (7). An
alternative 15N-NMR investigations shows no substantial
difference in tautomeric form between the neutral and
anionic form of 5-HOdC, with the amino form being the
only detectable form (9). The sensitivity of this method,
however, might not be high enough to detect alternative
tautomeric forms in the below 1% range. Although the
tendency of increased imino tautomeric form at higher
pH and higher temperature fits the trend of our observa-
tions it seems unlikely that its extent under any conditions
applied would be detectable in a UV-melting curve.
It is known that 5-HOrC is a redox active nucleoside
which can be oxidized with agents such as Fe3+ to
5-oxo-cytidine (5-OrC+, Figure 6, bottom) (25).
Therefore the question arises whether this oxidized form
of 5-HOrC is associated with the unusual melting
behavior. We rule out this hypothesis primarily based
on the reason that the conditions during nbnOrC
deprotection and subsequent manipulations for melting
curve measurements are not oxidizing, and that the
hydrogen bonding pattern of 5-OrC+ is expected to be
tautomerically stable and identical to that of a natural
rC unit which would not explain the biphasic transition.
We also questioned whether temperature-dependent
changes in the syn/anti ratios (syn at low and anti at
high temperatures) of the glycosidic torsion angle in
5-HOrC could be a reason for the biphasic melting tran-
sition. This cannot be ruled out completely but seems
less likely. Given that the hydroxy group is on C-5 and
not C-6, it is difficult to envisage a structural or
stereoelectronic force that would drive the base in syn
conformation which is generally the less preferred con-
formation for pyrimidine nucleosides. In addition, the
related base 5-nbnOrC where the bulky aryl group could
lead to steric interactions with the phosphodiester
backbone in the anti conformation, required for pairing
with G, behaves as rC and does not show biphasic melting
behavior. Also, the ab initio calculations of 5-HOdC did
not give any indications on a predominance of the
syn-conformer, neither in the neutral nor in the ionized
form (9).
Figure 6. Top: relevant tautomeric forms of 5-HOrC; bottom: the two
known redox states of 5-HOrC.
replaced by 5-nbnOrC clearly demonstrates that either the
ionization state of this group or the presence or absence of
a proton in this position significantly influences its base
recognition properties.
Several hypotheses can be formulated to explain this
biphasic melting behavior. One of these invokes a
chemical deamination from 5-HOrC to 5-HOrU under
the conditions of Tm-analysis. This can be disproved
based on the fact that the Tm profiles of duplexes contain-
ing authentic 5-HOrU in place of 5-HOrC are significantly
different from each other (Table 1). We also determined
the stability of nucleoside 2 at neutral pH and 80ꢀC over
24 h and found <5% 5-HOrC to 5-HOrU conversion by
NMR. Taken all together, deamination cannot be the
cause of the biphasic melting behavior. On another note
the results on the stability of 2 are in agreement with
earlier experiments on the oxidation of poly d(C-G) in
which it was shown that deamination occurs exclusively
on the level of the dihydroxylated dCg (Figure 1) but not
on 5-HOdC (6). Thus, most of the mutagenicity (C!T
transitions) seems to be due to the intermediate dCg and
must correlate with the rate of hydrolysis of the amino
group relative to the rate of dehydration to 5-HOrC of
the dCg intermediate.
Yet another, and probably more plausible, explanation
could be that the ionization state of the 5-OH group
influences indirectly the glycosidic torsion angle of the
nucleoside when incorporated into a DNA or RNA
backbone. The presence of a 50-phosphate unit is known
to increase the pKa of 5-HOrC by 1.1 units to 8.5, relative
to the free nucleoside. This increase has been associated
with electrostatic repulsion of the base oxyanion with the
negatively charged 50-phosphate unit (9). On the other
hand, ab initio calculations on 5-HOdCMP have shown
that in the neutral form the 5-OH group can form a strong
An obvious and tempting hypothesis arises from
invoking changes in the tautomeric equilibrium of
5-HOrC, either depending on the protonation state of
the 5-OH group or on temperature. It has been shown
before that 5-HOdC can besides the amino form also
adopt a minor imino tautomeric form (Figure 6, top).

Nucleic Acids Research, 2011, Vol. 39, No. 21 9431
H-bond with a 50-phosphate oxygen. It may therefore well
be that this hydrogen bond drives the glycosidic torsion
angle in a geometry which is unfavorable for correct base
pair formation with G within an oligoribonucleotide
duplex. Upon thermal denaturation of this H-bond
during heating the glycosidic bond could relax, thus
enabling efficient G-base recognition. Such a process
could be a reason for the observed double transition in
the melting curves. The local structural reorganizations
are likely to be too small to be detectable by CD spectros-
copy, explaining the almost identical CD spectra of
duplexes containing either a 5-HOrC-rG or a rC-rG
base pair.
We currently favor this last hypothesis but we are aware
that there may exist alternative explanations yet to be
discovered. For this, however, more biophysical data
including high resolution structural data on 5-HOrC
containing duplexes is needed. We note that besides tauto-
meric variability such structural effects could also deter-
mine the differences in the coding properties of 5-HOrC
units during RT.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
We thank Dr Alessandro Calabretta for providing the Tm
data of the duplex containing 5-HOrU (Table 1).
FUNDING
Funding for open access charge: University of Bern and
Swiss National Science Foundation (grant. No. 200020-
130373).
Conflict of interest statement. None declared.
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