Antiviral effect of ribonuclease conjugated oligodeoxynucleotides targeting the IRES RNA of the hepatitis C virus

Article abstract of DOI:10.1016/j.bmcl.2009.04.139

Hepatitis C virus (HCV) translation initiation is mediated by a highly structured and conserved RNA, termed the Internal Ribosome Entry Site (IRES), located at the 5′-end of its single stranded RNA genome. It is a key target for the development of new antiviral compounds. Here we made use of the recently developed HCV cell culture system to test the antiviral activity of artificial ribonucleases consisting of imidazole(s) linked to antisense oligodeoxynucleotides targeting the HCV IRES. Results from the cell culture model indicate that the naked antisense oligodeoxynucleotide displayed an efficient antiviral activity. Despite the increased activity observed with the addition of imidazole moieties when tested with the cell-free system, it appears that these improvements were not reproduced in the cellular model.

Full text of DOI:10.1016/j.bmcl.2009.04.139

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3581–3585
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antiviral effect of ribonuclease conjugated oligodeoxynucleotides targeting
the IRES RNA of the hepatitis C virus
Carly Gamble a,d, Maud Trotard b,d,e, Jacques Le Seyec b,d,e, Valérie Abreu-Guerniou a,c,d
,
Nicolas Gernigon c, Fabienne Berrée c, Bertrand Carboni c, Brice Felden a,d, Reynald Gillet a,d,
*
a Université de Rennes 1, UPRES JE 2311, INSERM U835, Biochimie Pharmaceutique, 2, Avenue du Prof. Léon Bernard, 35043 Rennes, France
b INSERM U522, Hôpital Pontchaillou, Avenue Henri Le Guilloux, 35033 Rennes, France
c Université de Rennes 1, UMR CNRS 6226, Sciences Chimiques de Rennes, Campus de Beaulieu, 35042 Rennes Cedex, France
d IFR 140 Génomique Fonctionnelle Agronomie et Santé, Université de Rennes 1, France
e Equipe Associée SERAIC, Université de Rennes 1, Rennes, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Hepatitis C virus (HCV) translation initiation is mediated by a highly structured and conserved RNA,
termed the Internal Ribosome Entry Site (IRES), located at the 50-end of its single stranded RNA genome.
It is a key target for the development of new antiviral compounds. Here we made use of the recently
developed HCV cell culture system to test the antiviral activity of artificial ribonucleases consisting of
imidazole(s) linked to antisense oligodeoxynucleotides targeting the HCV IRES. Results from the cell cul-
ture model indicate that the naked antisense oligodeoxynucleotide displayed an efficient antiviral activ-
ity. Despite the increased activity observed with the addition of imidazole moieties when tested with the
cell-free system, it appears that these improvements were not reproduced in the cellular model.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 27 March 2009
Revised 27 April 2009
Accepted 27 April 2009
Available online 4 May 2009
Keywords:
Hepatitis C virus
Imidazole
Antisense oligodeoxynucleotides
Chronic hepatitis C virus (HCV) infection affects more than 170
million people worldwide. The standard treatment consists of a
combination of pegylated interferon-alpha and ribavirin. However,
although such treatment is somewhat successful on HCV geno-
types 2 and 3, it is less effective with genotypes 1 and 4. This lack
of activity, the existence of some severe adverse side effects,
contraindications and the emergence of HCV drug resistance limit
efficacy and urge for the development of new therapies.1 Among
the therapeutic targets of HCV, its genomic RNA 50-untranslated
region (50-UTR) appears to be a good candidate for molecular ther-
apy. HCV has evolved an unusual mode of translation initiation
that is mediated by a highly structured RNA, termed Internal Ribo-
some Entry Site (IRES), located at the 50-end of its 9.5-kb nucleotide
RNA genome.2 Contrary to the canonical cap-dependent initiation
of most eukaryotic mRNAs, the HCV IRES can recruit 40S ribosomal
subunits independently of either an m7G-cap structure at the
mRNA 50-end or the scanning of the viral mRNA. This allows sim-
plified and efficient translation of its genomic RNA.3 The conserved
secondary structure of the HCV IRES comprises ꢀ340 nucleotides,
encompassing three main structural domains II to IV (Fig. 1).
The two large helical domains termed II and III are bridged by a
pseudoknot to domain IV that is a short stem-loop. Domain III
associates with the outer surface of the 40S ribosomal small sub-
unit platform and is required for the high affinity interaction of
the IRES with the latter, while domain II contacts the head of the
ribosomal subunit. Both structural domains interact with the mul-
tisubunit initiation factor eIF3.4 Among the complex structured
regions of the HCV IRES, subdomain IIId adopts the well character-
ized loop E motif. It plays an important role during the early steps
of ribosome recognition and therefore represents a potential target
for structure-based drug design.5Accordingly, aptamers targeted to
domain IIId were generated by in vitro selection (SELEX) and tested
both in a cell-free system and in cell culture. This was done using
transient transfection and a reporter mRNA where translation was
under the control of the HCV IRES.6 Antisense molecules targeting
the IRES can only prevent the translation of the HCV polyprotein. In
order to improve their activity, we recently developed a strategy
which combines the binding affinity of the antisense oligodeoxy-
nucleotides (AS-ODNs) with the capability of artificial ribonucle-
ases to cleave the RNA at specific locations.7 Conjugates were
synthesized by coupling imidazole moieties, known to mimic
RNase A catalytic centers,8 to AS-ODNs whose sequences were in-
spired by RNA aptamers targeting domain IIId.6 Therefore, these
compounds should disrupt the HCV life cycle by preventing trans-
lation and replication. In the present study we investigated the
toxicity and antiviral activity of the most efficient of these
* Corresponding author at present address: UMR CNRS 6026 Equipe Structure et
Dynamique des Macromolécules, Campus de Beaulieu 35042 RENNES Cedex,
France. Tel.: +33 2 23 23 45 07; fax: +33 2 23 23 50 52.
E-mail address: reynald.gillet@univ-rennes1.fr (R. Gillet).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.139

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C. Gamble et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3581–3585
microporated into Huh7.5.1 cells 17 h prior to infection with
U
U
IIIb
G
C
G
HCVcc. The ability of the oligodeoxynucleotides to interfere with
establishment of HCV replication was then measured relative to
that obtained with the NS-ODN, consisting of a randomly scram-
bled sequence of the AS-ODN. The growth and viability of the cells
was not affected by any of the oligodeoxynucleotides.11
U
A
U
U
U
U
C
A
C
U A
G C
190
G
C
G C
C G
The presence of the naked AS-ODN, the 50-imidazole or the 30-
imidazole conjugates within the cells reduced, in a dose-dependent
manner and up to 50%, the level of viral replication established in
Huh7.5.1 after their infection with HCVcc. The maximum antiviral
activity was achieved at a 100 nM concentration after 20 h of infec-
tion. No further inhibition was achieved by increasing the concen-
tration of oligodeoxynucleotides to 1000 nM (Fig. 3). Unlike the
results obtained with the cell-free system,7 the addition of an imid-
azole moiety to either the 30 or 50 end of the naked AS-ODN did not
result in an increase in their antiviral properties when tested on
the HCVcc propagation system. This result suggested that in cells,
the process of cleavage by a single imidazole was not the main fac-
tor responsible for activity as was previously observed in the cell-
free system.
To better mimic the catalytic center of RNase A, we prepared a
novel compound containing two imidazole moieties. The design
and synthesis of such bifunctional catalysts have received consid-
erable attention,12 since residues His 12 and His 119 in the cata-
lytic site of RNase A serve respectively as base and acid catalysts
and the protonated side chain of Lys-41 stabilizes the pentacoordi-
nated phosphorous intermediate during transition state.13 The bis-
imidazole-containing AS-ODN was synthesized by standard
protocol from the acid 8 (Scheme 1). First, urocanic acic 1 was
esterified with methanol14 and protected as its N-dimethylsulfa-
moyl derivative 3. Reduction with diisobutylaluminium hydride
and treatment with thionyl chloride afforded the allylchloride
5.15 Sodium salt of diethylmalonate was then alkylated with two
equivalents of 5 to give the diester 6, which was hydrogenated in
the presence of Pd on charcoal. Deprotection and decarboxylation
were simultaneously achieved by heating at reflux in 6 M HCl.
Analysis of the reaction mixture revealed the concomitant forma-
tion of dimethylamine hydrochloride. Final purification by ion
exchange chromatography on Dowex H+ 50W8 gave the desired
compound 8.16 The coupling reaction was carried out in the
presence of PyBOP, HOBT and N-methylmorpholine in DMF at
the 50-end of the oligodeoxynucleotide, since the 50 mono-imidaz-
ole displays the highest activity in vitro.7,17
First, the antiviral activity of this bis-imidazole compound was
tested in a cell-free system and compared to the naked antisense
and to the mono-imidazole compounds. The specific cleaving
activity of the bis-imidazole coupled to the AS-ODN was also con-
firmed by using an in vitro transcribed and labeled HCV IRES RNA
fragment. The inhibitory effects of the bis-imidazole conjugate on
HCV IRES driven translation of a Renilla luciferase reporter mRNA
were also confirmed by using a reticulocyte based translation
assay. However, despite the use of two imidazole groups within
the same molecule, a similar level of viral translation inhibition
was achieved as compared to the 50-mono imidazole compound,
whatever the concentration used (not shown).
We then decided to investigate the action of this modified oligo-
deoxynucleotide in a more physiological system by using hepatic
cell cultures infected with HCVcc. The optimal concentration deter-
mined in the previous experiments (100 nM) was the concentra-
tion chosen to test the bis-imidazole conjugate. As observed with
the cell-free approach, there was no improvement in the antiviral
activity of the AS-ODN when coupled to two imidazoles instead
of one. Indeed, whatever the compound (naked or coupled AS-
ODN) previously introduced into the cell, the establishment of viral
replication after HCVcc infection was inhibited by approximately
half (Fig. 4).
C
C
A U
G C
A
C
A
A
U
G
G C
G C 240
A U
C
C G
C G
G A
C
G
A
A
80
U
U
C
G
U G
U A
U
G
C
G
A
G
C
G
U
U
IIIa
A U
A U
A
IIIc
U
U
G
CACCGG
GUGGCC
II
C
A
A
GGG
CCC
G
U
A
G
U
A
A
A
A
G
G C
G
A U
C G
G A
C G
A U
C G
G C
C G
G C
150
G
100
U A
C G
U
A
U
U
C
G C
U
IIId
G
U
U
G
U
G
C
G C
A U
U
C
A
U
G
U
G
U
A
AG
UU
UAGUG
AGCGC
GCCG
CGGA
G
C
C
A
G
A
UG
A
A
C
U
G
G
G
A
C
C
U
C
U
G
A
C
C
G
50
U
G
G
U
G
U
286
G C
U A
C G
C G
A
A
IIIe
C
U
G
A
5’
G
A
U
GCCU
UGGG
ACCCCCCCUCCCGGG
GGAGGGCCC
CUCC
A
CG
GC
U
C
U
U
U
U
G
C
A
G
GC
G
3’
U
CUAAACCU
A
C
G C
A U
A
A
C G
G C
U A
G C
C G
IV
A
A
340
G
U
U
C
A
Figure 1. Secondary structure of the IRES of the HCV genomic RNA. The predicted
binding site of the 17-mer antisense oligodeoxynucleotide AS-ODN used in this
study within the domain IIId sequence is indicated (dashed).
compounds in vitro by making use of the recently developed prop-
agation system of infectious HCV particles in cell culture (HCVcc).9
This system reproduces the entire life cycle of the virus in a hepatic
cell line. In order to improve their activity, a new original com-
pound containing two imidazole moieties instead of one was also
designed, produced and assessed.
To facilitate screening of the compounds, we used a modified
HCVcc with a genome that contains a luciferase reporter gene.10
The naked antisense DNA (AS-ODN) and non specific DNA (NS-
ODN) sequences used for this study were as follows: ACCCAACAC-
TACTCGGC and CAACCCTAGCCCGTCAA respectively. In the first
series of experiments, the naked AS-ODN or the AS-ODN coupled
with either 50-imidazole or with 30-imidazole7 (Fig. 2) were

C. Gamble et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3581–3585
3583
NH2
N
NH
2
N
N
N
N
O
O
N
O
P
O
O
O
N
N
H
O
O
O
ACCCAACACTACTCGG
N
H
O
O
P
H
N
CCCAACACTACTCGGC
O
O
H
O
N
N
O
3’ imidazole AS-ODN
5’ imidazole AS-ODN
NH2
HN
N
N
N
O
N
N
O
P
O
N
H
O
O
N
O
O
HN
CCCAACACTACTCGGC
5’ bis-imidazole AS-ODN
Figure 2. Sequences of ribonuclease conjugated oligodeoxynucleotides used in this study.
140
NS AS 5'-imidazole 3'-imidazole
120
100
80
60
40
20
0
10
25
50
100
1000
Concentration (nM)
Figure 3. Effect of naked or mono-imidazole coupled oligodeoxynucleotides on cell infection by HCVcc. Huh7.5.1 cells were electroporated with either the non-specific ODNs
(NS-ODN), the naked antisense ODN (AS-ODN), the 50-imidazole or the 30-imidazole conjugated AS-ODN at different concentrations prior to infection with HCVcc.
Experiments were performed in triplicate and error bars represent the standard deviation of the mean. Results were normalized against cell number as determined by WST-1
Cell Proliferation Assay (Roche). Values are expressed as percentages of the NS controls.
COOH
COOMe
COOMe
DiBAL, CH Cl
2
N
N
N
N
OH
N
N
1) NaH, THF
2
MeOH, H+
81%
2) Me NSO Cl
N
H
65%
N
H
2
2
SO NMe
2
SO NMe
2
2
2
61%
4
3
2
1
Me NO S
Me NO S
2 2
2
2
N
N
SOCl ,
2
Cl
N
N
N
N
N
H , Pd/C
diethyl malonate
NaH, THF, 47%
2
COOEt
COOEt
COOEt
COOEt
99%
CH Cl
2
2
N
SO NMe
2
2
67%
N
N
6
7
Me NO S
Me NO S
2
2
2
2
5
NH
N
2
HN
N
HN
HN
N
+
PyBOP, HOBT,
1/ H , Δ
N
N
O
N
N
N
O
O
COOH
2/ Dowex H+
75%
N-methylmorpholine,
Oligonucleotide
N
H
O
P
O
O
HN
O
CCCAACACTACTCGGC
5’ bis-imidazole AS-ODN
Scheme 1. Synthesis of the bis-imidazole conjugated AS-ODN.
8

3584
C. Gamble et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3581–3585
120
100
80
transfection of the oligodeoxynucleotides was possible using
NS
AS
Bis-imidazole
lipofection, we then tested the action of the oligodeoxynucleotides
on Huh7.5.1 cells that were already infected with HCVcc. As seen in
Figure 5, ꢀ10-fold higher concentration of oligodeoxynucleotides
was required to detect antiviral activity when HCV replication
was already established in cells. A maximum of 50% viral inhibition
was achieved at a concentration of 1000 nM. The limited level of
inhibition achieved in these experimental conditions may be partly
ascribed to the eventual competition of the compounds with cellu-
lar factor(s) interacting with the same region of the HCV RNA. It
may also be due to the higher level of IRES RNAs to target. How-
ever, no increase in inhibition was achieved with the addition of
imidazole or bis-imidazole to the naked ODN sequence.
60
40
20
0
100
Concentration (nM)
Noteworthy, the same level of inhibition of viral replication was
achieved when comparing a 50-end imidazole linked 20-O-Me-RNA
oligonucleotide to its naked counterpart (data not shown).
Figure 4. Effect of the bis-imidazole oligodeoxynucleotide conjugate on cell
infection by HCVcc. Huh7.5.1 cells were electroporated with either the non-specific
ODNs (NS-ODN), the naked antisense ODN (AS-ODN) or the newly synthesized
oligodeoxynucleotide, 50bis-imidazole, at a concentration of 100 nM. Experiment
was performed in triplicate and error bars represent the standard deviation of the
mean. Results were normalized against cell number as determined by the WST-1
Cell Proliferation Reagent (Roche). Values are expressed as percentages of the NS
controls.
Therefore, despite the increased activity observed with the
addition of imidazole moieties when tested with a cell-free trans-
lation assay, it appears that this improvement is not observed in
infected Huh7.5.1 cells. We cannot rule out that factor(s) present
in human hepatic cells, but absent from rabbit reticulocytes, could
outweigh or mask the cleavage sites targeted by the imidazole
moieties. Therefore, in order to improve their intrinsic effects,
stabilized in vivo compounds will be developed. A promising
approach could be the use of short peptide nucleic acids (PNA) that
have recently been shown to display stronger activity than DNA or
RNA when targeting HCV IRES driven translation.18 Furthermore,
the use of a peptidic bond to bind the RNAse A mimic to AS-ODNs
makes this system adaptable and easily interchangeable with other
cleaving agents. The tool developed here will now be adapted to
target other domains of the genomic RNA of HCV.
One hypothesis for this lack of improvement might be the
extended time the oligodeoxynucleotides were present in the cells
before addition of HCVcc (17 h). Such a prolonged infection period
may lead to a partial inhibition of the cleaving properties of the
compounds. Infecting cells containing oligodeoxynucleotides at
time-points of less than 17 h was attempted. However, this proved
to be toxic to the cells, most likely because they have not recovered
from transfection.
We therefore decided to test our oligodeoxynucleotide panel by
infecting cells with HCVcc prior to transfection. This would allow
the oligodeoxynucleotides to act on the IRES immediately, rather
than after a 17 h delay where degradation may take place. The
trypsination of Huh7.5.1 cells after infection with our recombinant
HCVcc decreased the proportion of HCV replicating cells. Since it is
a step required for microporation, we therefore chose to transfect
Huh7.5.1 cells after HCVcc infection using a lipid reagent.
We initially confirmed that the antiviral activity of the AS-ODN
could be achieved using lipofection. Thus, AS-ODN was transfected
at a final concentration of 100 nM, the concentration which gives
the maximum levels of inhibition with microporation. After a
17 h incubation period, cells were infected with HCVcc. Using lip-
ofection yielded similar inhibition results to those obtained with
microporation. As found previously, there was no cytotoxic effect
of the oligodeoxynucleotides. Once we had confirmed that
Acknowledgments
This work was supported by grants from the ‘Région Bretagne’
(PRIR Grant No. 691) and from the ‘Ligue Nationale contre le
Cancer (Coordination grand-ouest)’. C.G. was supported by a post-
doctoral fellowship from the University of Rennes 1; V.G. was
supported by a post-doctoral fellowship from the ANRS (Agence
Nationale de Recherche sur le SIDA et les Hépatites Virales’, and
M.T. and N.G. were recipients of a fellowship from the ‘Région
Bretagne’. We would like to thank Dr. Francis Chisari from The
Scripps Research Institute (TSRI) for the Huh7.5.1 cells; Dr Charles
M. Rice for the construct FL-J6/JFH-5/C19Rluc2AUbi and Dr. Pascal
Loyer, INSERM U522, ‘Plateforme Vectorisation non virale IBISA
Ouest-Genopole’ for the use of the Microporator (LabTech France).
140
NS
AS
5'-imidazole
3'-imidazole
bis-imidazole
120
100
80
60
40
20
0
10
100
Concentration (nM)
1000
Figure 5. Effect of oligodeoxynucleotides on cells already infected with HCVcc. Huh7.5.1 cells were initially infected with HCVcc. After a 17 h incubation, cells were
transfected with either the non-specific ODN (NS-ODN), the naked antisense ODN (AS-ODN), the 50-imidazole, the 30-imidazole or the 50bis-imidazole at different
concentrations. Experiments were performed in triplicate and error bars represent the standard deviation of the mean. Results were normalized against cell number as
determined by WST-1 Cell Proliferation Reagent (Roche). Values are expressed as percentages of the NS controls.

C. Gamble et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3581–3585
3585
2004, 32, 3887; (d) Fouace, S.; Gaudin, C.; Picard, S.; Corvaisier, S.; Renault, J.;
Carboni, B.; Felden, B. Nucleic Acid Res. 2004, 32, 151–157.
Supplementary data
13. Breslow, R. Acc. Chem. Res. 1991, 24, 317.
14. Pirrung, M. C.; Pei, T. J. Org. Chem. 2000, 65, 2229.
15. He, Y.; Chen, Y.; Wu, H.; Lovely, C. J. Org. Lett. 2003, 3623.
Supplementary data (cell culture, production and titration of
HCVcc, Huh7.5.1 cell transfection methods and cell viability and
luciferase assays) associated with this article can be found, in the
online version, at doi:10.1016/j.bmcl.2009.04.139.
16. 5-(1H-imidazol-4-yl)-2-[3-(1H-imidazol-4-yl)propyl]pentanoic acid 8: 200 mg of
6 (0.34 mmol) were dissolved in 10 mL of methanol. 21 mg of Pd/C 10% were
added to the solution. After 20 h of stirring under 45 bars of dihydrogen, the
mixture was filtered and the filtrate concentrated to dryness to give 200 mg of
7 as a clear yellow oil (99% yield). The product was used without further
purification in the next step. 1H NMR (300 MHz, CDCl3) d 1.24 (t, 6H, J = 7.1 Hz),
1.50–1.56 (m, 4H), 1.92–1.98 (m, 4H), 2.60 (t, 4H, J = 7.4 Hz) 2.86 (s, 6H), 4.18
(q, 4H, J = 7.1 Hz), 7.00 (s, 2H), 7.85 (s, 2H). 13C NMR (75 MHz, CDCl3) d 171.4,
143.1, 136.1, 113.7, 61.2, 57.1, 38.1, 31.6, 27.9, 23.1, 14.0. 200 mg of crude 7
were dissolved in 10 mL of 6 M HCl. The solution was refluxed for 4 days before
being allowed to cool to room temperature. Evaporation to dryness afforded an
oily residue. 1H NMR (300 MHz, D2O) d 1.48–1.53 (m, 8H), 2.28–2.39 (m, 1H),
2.59–2.73 (m, 4H), 7.07(s, 2H), 8.42 (s, 2H). 13C NMR (75 MHz, D2O) d 180.9,
133.4, 132.6, 115.1, 44.8, 30.6, 25.4, 23.5. Dimethylamine hydrochloride was
identified in the crude mixture by its NMR data. 1H NMR (300 MHz, D2O) d
2.59 (s, 6H). 13C NMR (75 MHz, D2O) d 34.5 (same values than those obtained
with an authentic sample). The residue was then dissolved in 15 mL of distilled
water before purification by ion-exchange column chromatography Dowex H+
50W8. The product was eluted with concentrated ammonium solution. The
fractions were pooled, concentrated and lyophilized to give 70 mg of 8 as a
very hygroscopic clear brown solid (75% yield). 1H NMR (300 MHz, D2O) d
1.29–1.34 (m, 4H), 1.43–1.53 (m, 4H), 2.06–2.16 (m, 1H), 2.56 (t, 4H, J = 7.0 Hz),
6.98 (br s, 2H), 8.17 (br s, 2H). 13C NMR (75 MHz, CDCl3) d 185.1, 135.1, 133.5,
115.8, 48.5, 31.9, 26.2, 24.1. ESI (MeOH) HRMS [M+H]+ calculated for
C14H21N4O2 277.16645; found. 277.1658.
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17. The phosphodiester oligodeoxynucleotide conjugate, prepared as previously
described (Ref. 7), was analyzed and purified by reverse phase HPLC on
Uptisphere ODB C18-5
l columns (Interchim, France) using Agilent 1200
10. Tscherne, D. M.; Jones, C. T.; Evans, M. J.; Lindenbach, B. D.; Mckeating, J. A.;
Rice, C. M. J Virol. 2006, 80, 1734.
11. For detailed methods see Supplementary data.
12. (a) Verbeure, B.; Lacey, C. J.; Froyen, M.; Rozenski, J.; Herdewijn, P. Bioconjugate
Chem. 2002, 13, 333; (b) Oivanen, M.; Kuusela, S.; Lonnberg, H. Chem. Rev. 1998,
98, 961; (c) Beloglazova, N. G.; Fabani, M. M.; Zenkova, M. A.; Bichenkova, E. V.;
Polushin, N. N.; Sil’nikov, V . N.; Douglas, K. T.; Vlassov, V. V. Nucleic Acid Res.
system (Agilent). The purified oligodeoxynucleotide conjugate was analyzed
by MALDI-TOF mass spectrometry using an UltraFlex Mass Spectrometer
(Bruker Daltonics, Germany). The measured mass was in accordance to the
theoretically calculated value.
18. Alotte, C.; Martin, A.; Caldarelli, S. A.; Di Giorgio, A.; Condom, R.; Zoulim, F.;
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