Biological and physicochemical characterization of siRNAs modified with 2',2'-difluoro-2'-deoxycytidine (gemcitabine)

Article abstract of DOI:10.1039/b9nj00746f

The use of synthetic short interfering RNAs (siRNAs) is currently a method of choice to manipulate gene expression in mammalian cells. Efforts aimed at improving siRNA biological activity, including increased silencing properties, higher substrate specifi

Full text of DOI:10.1039/b9nj00746f

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PAPER
www.rsc.org/njc | New Journal of Chemistry
Biological and physicochemical characterization of siRNAs modified
with 20,20-difluoro-20-deoxycytidine (gemcitabine)wz
Malgorzata Sierant,* Milena Sobczak, Magdalena Janicka, Alina Paduszynska
and Danuta Piotrzkowska
Received (in Montpellier, France) 10th December 2009, Accepted 15th January 2010
First published as an Advance Article on the web 2nd March 2010
DOI: 10.1039/b9nj00746f
The use of synthetic short interfering RNAs (siRNAs) is currently a method of choice to
manipulate gene expression in mammalian cells. Efforts aimed at improving siRNA biological
activity, including increased silencing properties, higher substrate specificity and cellular stability,
lower cytotoxicity, and improved target delivery, have been made through the introduction of
various chemical modifications into the siRNA strands. In these studies, we present the synthesis
of oligoribonucleotides with the single replacement of a cytidine unit for 20,20-difluoro-20-deoxycytidine
(gemcitabine, dFdC) and the use of them in a series of siRNAs for gene silencing experiments.
The dFdC modifications are located in six different positions of the antisense strand, which are
crucial for siRNA silencing activity. The results indicate a position-dependent tolerance for the
dFdC modification. Gemcitabine units present in the ‘‘seed region’’, at positions 1 or 8, resulted
in only a B15% silencing activity in the corresponding duplexes. The dFdC unit at position
10 virtually switched off the silencing activity (below 10%), while the dFdC unit at the positions
2, 4 or 5 produced duplexes of silencing potential comparable to that of the non-modified duplex
(70% silencing). The dFdC modification had little impact on the structure of the siRNA duplexes,
as determined by circular dichroism analysis, while melting experiments showed their lower
thermal stability.
molecule, after introduction into the cell, is complexed
1. Introduction
by a nucleoprotein effector complex RISC (RNA-induced
RNA interference (RNAi) is a powerful biological process for
silencing complex). Then, one of the two strands works as
the sequence-specific silencing of gene expression in diverse
the guide strand and directs the complex to a complementary
eukaryotic cells. RNAi has tremendous potential for
sequence of the target mRNA, while the other one is endo-
nucleolytically degraded during the assembly process.11,12 The
functional genomics and the development of novel gene-
specific therapies.1–3 Specific gene silencing may be induced
core of the effector complex contains one of the Argonaute
by synthetic short interfering RNAs, which are 21–23-nucleotide
(Ago) proteins, which are members of the PAZ/PIWI
family.13,14 Depending on the nature of the Ago protein and
(nt) long duplexes consisting of sense (non-guide, passenger)
and antisense (guide) complementary strands, with two
on the degree of complementarity between the siRNA and the
unpaired nucleotides (2-nt overhangs) at each 30-end (Fig. 1).
target sequence, the association of human RISC with mRNA
SiRNA duplexes are designed to mimic the RNase III
results in different silencing modes, as endonucleolytic
processing intermediates of naturally-expressed dsRNAs, such
cleavage, translational repression, deadenylation or sequestra-
as miRNAs, to effectively enter the RNAi pathway.4–7
tion to the P-body compartments.15 Among the four Ago
Naturally processed siRNAs or miRNAs are characterized
proteins coded in the human genome (hAgo1-4), only hAgo2
by the presence of 50-phosphates and 30-terminal overhangs,
is endonucleolytically active.14,16,17 Ago proteins are composed
which are important for their biological activity.8,9 Synthetic
of four functional domains: amino-terminal N, PAZ, and
siRNA duplexes with 50-hydroxyl ends are rapidly phosphorylated
C-terminal MID and PIWI. The catalytic site of the slicer is
inside cells by the cellular kinase Clp1.10 An siRNA duplex
situated at the PIWI domain, the MID domain recognizes and
anchors the 50-phosphate of the guide strand, whereas the
2-nt overhang at the 30-end is recognized by the PAZ
Centre of Molecular and Macromolecular Studies,
Polish Academy of Sciences, Lodz 90-363, Sienkiewicza 112.
E-mail: msierant@bio.cbmm.lodz.pl; Fax: +48 42 681 54 83;
Tel: +48 42 680 32 48
w This paper is dedicated to Professor Wojciech J. Stec on the occasion
of his 70th Birthday. This article is part of a themed issue on
Biophosphates.
z Electronic supplementary information (ESI) available: Silencing
activity of siRNAs (1 nM) in HeLa cells; cytotoxicity of siRNA
duplexes in HeLa cells; nucleolytic stability of siRNA duplexes in
10% FBS. See DOI: 10.1039/b9nj00746f
domain.18–21 Ago-mediated target mRNA cleavage requires
Watson–Crick base pairing between the guide strand of
siRNA and the target strand, spanning both the seed region
(positions from 2nd to 8th nucleotide) and the cleavage
site (phosphate group opposite to the internucleotide linkage
located between 10th and 11th nucleotide, counted from
the 50-end of the guide strand).4 Recently, Wang et al.
reported structural studies that reveal the molecular dynamics
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Fig. 1 Short interfering RNA (siRNA) structure with indicated sites crucial for silencing activity, which represent potential positions for chemical
modification.
of Thermus thermophilus Ago protein as it binds to and slices
its RNA target.19,22,23
modified siRNAs were compared to those of the parent,
non-modified (NM) duplex. We did not expect any significant
structural changes of the RNA duplexes carrying the dFdC
modification, as the structural features of gemcitabine fit
well to the A-type RNA conformation.40 Rather, we were
interested in to what extent a single gemcitabine unit present in
the ‘‘seed region’’ or in the cleavage site modulates the activity
and thermodynamic stability of the corresponding siRNA
duplexes.
Along with efforts aimed at explaining the mechanism of the
RNA gene silencing process,18–23 tremendous emphasis has
been placed on the improvement of the most important
features of siRNAs: (i) silencing potency, (ii) cellular uptake,
(iii) nucleolytic stability, (iv) target specificity and (v) elimination
of immune and non-specific ‘‘off-target’’ effects. A possible
approach to reach this goal is the introduction of chemical
modification into the duplex.24–26 A variety of chemical
modifications have been proposed and have led to a significant
growth in understanding of the mechanism of RNAi. Widely
used in siRNAs are modifications of the ribose moiety;
especially extensively modified is the 20-OH group.27–32 Chiu
and Rana, in one of the earliest studies on chemically modified
siRNA, have shown that, while preservation of the A-form
duplex structure is important for its silencing activity, the
20-OH group is not required.28 In general, 20-modifications are
more or less tolerated, depending on the number and position
in siRNA strands. Functional siRNAs can contain fluorine at
the 20-position, e.g., 20F-RNA is one of the best known
siRNA modifications, and several replacements are tolerated
throughout the sense and antisense strands.28,29,33 Such
modifications strongly favour the A-form helical structure of
the duplex and significantly increase the stability of siRNAs in
serum.34 Another one, a 20F-ANA modification, with the
opposite stereochemistry of the fluorine (in the ara position),
developed as a DNA-mimic, is still well tolerated in siRNA
duplexes, including the fully modified sense strand and in
partial modifications of the antisense strand.35,36
2. Results and discussion
2.1 Selection of the model siRNA sequence and sites for dFdC
modification
In our previous studies, we designed several siRNAs for
target sequences of human/mouse/rat (h/m/r) beta-secretase
(BACE1) mRNA and validated their silencing activity in
cellular models.41 All of those siRNAs had a conventional
duplex structure: a 19-bp stem and 2-nt overhangs (TT) at the
30-ends. For the present studies, we chose the duplex h/m Gem
of moderate activity for the target sequences of human
(629–659) and mouse (602–632) BACE1 mRNA. Importantly,
in the antisense strand, the molecule Gem siRNA has several
cytidine units at positions considered important for silencing
activity. For the modification, we chose positions 1, 2, 4, 5, 8
and 10 (counting from the 50-end), and replaced the respective
cytidine units with a dFdC unit (Fig. 2). The 30-O-phosphor-
amidite monomer of the protected 20,20-difluoro-20-deoxycytidine
was prepared according to Scheme 1. All RNA oligo-
nucleotides (with and without gemcitabine substitution) were
synthesized in house using solid phase phosphoramidite
chemistry.42,43 After the routine synthesis, the oligonucleotides
were deprotected, purified and assembled into duplexes with
the non-modified sense RNA strand (details are given in the
Experimental section).
In the present study, we describe the synthesis of six
siRNA duplexes with
a
single modification in the
antisense strand, where a cytidine unit is replaced with
20,20-difluoro-20-deoxycytidine (dFdC, gemcitabine, IUPAC name:
4-amino-1-(2-deoxy-2,2-difluoro-b-D-erythro-pentofuranosyl)-
pyrimidin-2(1H)-one). Gemcitabine, an analog of deoxycytidine
(dC), is an anticancer drug that is widely used in the treat-
ment of prevalent human cancers.37,38 dFdC is also a potent
sensitizer to radiation, and this activity requires metabolic
transformation to the corresponding triphosphate (dFdCTP)
and incorporation of dFdCp into DNA.39 The modification
was introduced into several positions important for siRNA
activity. The gene silencing activity, thermodynamic
properties, nucleolytic stability and cytotoxicity of the
2.2 Silencing activity of dFdC-modified siRNAs
To investigate the silencing activity of siRNA duplexes, we
applied a dual fluorescence reporter system.44 This model
system is based on a measurement of the relative fluorescence
intensity of the enhanced green fluorescent protein (EGFP)
and the coral-derived (Discosoma spp.) red fluorescent protein
(RFP), expressed from plasmids delivered exogenously into
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Fig. 2 The sequences of siRNAs used in this study (with indicated dFdC-modified sites) and their silencing activity in HeLa cells (5 nM siRNAs),
presented as percentages of GFP expression of the control cells transfected with non-silencing control siRNA.
Scheme 1 A schematic representation of 20,20-difluoro-20-deoxycytidine phosphoramidite synthesis, RNA oligonucleotide preparation and
deprotection. More detailed information is included into the Experimental section.
HeLa cells. Two reporter plasmids were used: commercially
available pDsRed2-N1 (BD Biosciences) and pBACE1-GFP
all the dFdC-modified duplexes are less active. Duplexes
Gem2, Gem4 and Gem5, modified at positions 2, 4 and 5,
respectively, are remarkably active (50–60% silencing). In
contrast, dFdC modifications at positions 1, 8 and 10 are
detrimental to the activity of siRNA duplexes (Gem1, Gem8
and Gem10, respectively), of which the silencing effect is
below 20%.
plasmid coding
a fusion protein BACE1-GFP, kindly
provided by Dr Weihong Song (The University of British
Columbia, Vancouver, Canada).45 All transfection experi-
ments were performed with Lipofectamine 2000 (Invitrogene)
on a 96-well plate in a 48 h assay. After transfection of the
plasmids and of the screened siRNA (Fig. 2) (used in 1 or
5 nM concentrations) into HeLa cells, the cells were incubated
for 48 h, lysed and the relative fluorescence of GFP to RFP
determined. The results of the screening experiments are
shown in Fig. 2. While the parent, non-modified duplex
(NM) silences the expression of the target gene by ca. 70%,
2.3 Cytotoxicity of dFdC-modified siRNAs
Chemically modified RNA duplexes may induce numerous
cytotoxic effects.27,31 In the case of the screened Gem1–Gem10
siRNAs, the cytotoxic effects might originate from the nature
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of the introduced modified unit, as gemcitabine is the very
effective anticancer drug.37–40 Therefore, the influence of the
dFdC modification on cell viability was evaluated by screening
the toxicity of siRNAs Gem1–Gem10 for HeLa cells. The
siRNA duplexes were used at five chosen concentrations
(1, 5, 10, 20 and 50 nM) with an appropriate amount of
transfection reagent Lipofectamine 2000. Cell viability was
assessed by the MTT assay. Untreated HeLa cells were used as
a positive control. Virtually no toxic effect for any sample was
observed (ESI, Fig. S2z), and the cytotoxicities of non-
modified siRNA and the dFdC-modified duplexes were similar.
The biggest, but still minor, effect on cell viability (B10–15%
reduction) was observed for siRNAs at concentrations
above 20 nM.
Fig. 3 CD spectra of the dFdC-modified siRNA duplexes. Reagents
and conditions: 10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA buffer
(pH 7.4), temperature 25 1C, duplex concentration 2 mM.
2.4 Nucleolytic stability of modified siRNAs in 10% FBS
The increased resistance of siRNAs to endo- and exo-
nucleolytic cleavage is a primary factor that decides on the
basic features of RNAi, such as the efficiency of cellular
uptake, the duration of gene expression inhibition and the
dosing schedules required to achieve therapeutic effects. We
examined the stability of siRNA duplexes with dFdC
modification in 10% active fetal bovine serum (FBS). All the
duplexes (NM and Gem1–Gem10) (Fig. 2) were radioactively
labelled (32P) at the 50-end of the sense strand and treated with
10% FBS in RPMI medium (the same medium composition as
for HeLa cell culturing) at 37 1C. Aliquots were collected after
incubation for 0, 1, 5, 15, 30, 60, 120, 240, 360 min and 24 h,
and examined for the presence of the intact duplex by
native 20% polyacrylamide gel electrophoresis (PAGE). The
stability of modified siRNAs (Gem1–Gem10) in 10% FBS was
comparable to the stability of the reference NM duplex
(half-life longer than 6 h). The respective data are shown in
the ESI (Fig. S3).z Besides, the stability of the single-stranded
dFdC-containing oligonucleotides was also screened. All of
them were unstable in these conditions and were hydrolyzed
during the first minute, indicating that a single modification
within the chain does not prevent ssRNA from undergoing
endonucleolytic cleavage.
the positive Cotton effect at 268 nm and a shift of the
crossover point from 240 to 245 nm were observed, suggesting
that the structure of this duplex is distorted from the A-RNA
type (Fig. 3).
2.6 Hybridization studies
UV-monitored thermal dissociation profiles were determined
for all the dFdC-modified siRNAs and for the non-modified
duplex (Fig. 4). Melting temperatures (Tm) were calculated as
the maximum of the first derivative DA/DT for the thermal
dissociation profiles. The biggest difference in the thermal
stability (DTm = 6 1C) and, thus the most severe decrease in
stability of the modified duplex in comparison to the
non-modified congener, was observed for the Gem10 duplex,
with the dFdC modification in the central position of the
antisense strand. A similar decrease of Tm (B5 1C) was found
for Gem1, Gem2 and Gem8, with the dFdC modification at the
1st, 2nd and 8th position, counting from the 50-end of
the antisense strand. Based on the two-state van’t Hoff model,
the melting temperatures (Tm cal) and standard thermo-
dynamic parameters (DH1, DS1 and DG1) were calculated
(Table 1).46 From a thermodynamic point of view, the binding
affinity constant, Ka, is defined in terms of the free energy of
2.5 Analysis of the structure of the dFdC-modified duplexes
by circular dichroism
Circular dichroism (CD) spectra were collected for all the
dFdC-modified siRNA duplexes and compared with the CD
spectrum of the non-modified duplex. The majority of
CD spectra indicated a typical A-type structure of the
double-stranded RNA (a maximum of the positive Cotton
effect at 268 nm and a crossover point at 240 nm). The
obtained result was expected, based on the earlier report of
Konerding et al.,40 who determined the structural effect of the
dFdC substitution on model DNA of the Okazaki fragment.
Respective NMR studies revealed that the dFdC sugar
conformation differed from unmodified deoxycytidine and
adopted the C30-endo sugar ring puckering (RNA-like A-type
sugar ring conformation). While the majority of duplexes
adopted the typical A-type RNA structure, in the CD
spectrum recorded for duplex Gem4, with the dFdC substitution
at position 4, a significantly lower intensity of the maximum of
Fig. 4 UV-monitored thermal dissociation profiles for non-modified
duplex NM and siRNA duplexes with dFdC modifications. Oligo-
nucleotide concentration 2 mM, 10 mM Tris-HCl, 100 mM NaCl,
1 mM EDTA buffer (pH 7.4), dissociation at a temperature gradient of
1 1C minꢁ1
.
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Table 1 Melting temperatures and calculated thermodynamic parameters for the thermal dissociation of screened siRNA duplexes
Name
Tm exp/1C
Tm cal/1C
DH1/kcal molꢁ1
DS1/cal Kꢁ1 molꢁ1
DG1/kcal molꢁ1
NM
72.6
67.4
67.5
68.2
70.1
67.6
66.8
70.55
66.09
66.32
nd
68.22
65.42
64.84
ꢁ66.59
ꢁ68.18
ꢁ70.12
nd
ꢁ83.09
ꢁ73.18
ꢁ68.86
ꢁ164.92
ꢁ172.15
ꢁ177.73
nd
ꢁ214.56
ꢁ187.37
ꢁ174.91
ꢁ15.44
ꢁ14.79
ꢁ15.00
nd
ꢁ16.54
ꢁ15.08
ꢁ14.62
Gem 1
Gem 2
Gem 4
Gem 5
Gem 8
Gem 10
binding (DG1), Ka = exp(ꢁDG1/RT), where R is the gas
constant and T is the absolute temperature. The free energy
of binding is, in turn, defined by the enthalpy (DH1) and
entropy (DS1) changes and DG = DH1 ꢁ TDS1. Therefore
Ka = exp[ꢁDH1 ꢁ TDS1)/RT] = exp[ꢁDH1/RT] ꢁ exp[DS1/R].
The binding affinity can be enhanced by driving DH1 more
negative, by driving DS1 more positive or by a combination of
both effects. The binding enthalpy primarily reflects the
strength of the interactions of the siRNA strands in the duplex
(hydrogen bonds, base stacking, etc.) relative to those existing
with solvent molecules for the single strands. On the other
hand, the entropy change mainly reflects two contributions:
changes in solvation entropy and changes in conformational
entropy.47
Gemcitabine was moderately or poorly tolerated in
some positions of the ‘‘seed region’’ of the guide strand.
Modifications at positions 1 and 8 had a strong impact on
efficacy, while silencing activity was only ca. 15% (for com-
parison, 70% GFP silencing by the non-modified duplex).
A similar situation was observed for siRNA with gemcitabine
at position 10, where activity declined to below 10%. Gemci-
tabine substitution at the positions 2, 4 and 5 caused only
moderate changes in silencing efficiency in comparison to the
non-modified duplex. CD spectra for most of the screened
duplexes indicated a typical A-shaped RNA structure, but the
melting temperature parameters of the modified duplexes were
decreased by 5–6 1C.
From already published data, we know that gemcitabine
cytotoxicity is the consequence of an alteration in DNA
polymerase-mediated DNA synthesis following dFdCp
mis-incorporation. dFdC adopts an RNA-like ribose confor-
mation (C30-endo).40 In addition, the geminal difluoro group
of dFdC substantially alters the electrostatic surface of the
duplex at the site of substitution. Such substantial changes
may be potentially important for protein binding. Further-
more, the steric clashes may also contribute to a change in the
siRNA silencing activity, as difluorine substitution alters the
size of the modified moiety relative to the non-modified
moiety.40 We suppose that changes in the silencing activity
of the studied siRNAs originate from intrinsic gemcitabine
properties that are the consequence of a disturbance in
the RNA–protein interaction rather than in RNA–RNA
recognition.
Considering the melting profile recorded for Gem4, we
observed some pre-melting and a low hyperchromic effect;
so in this case, we could not calculate standard thermo-
dynamic parameters using the two-state van’t Hoff model.
Comparing the binding enthalpy of the non-modified (DH1 =
ꢁ66.59 kcal molꢁ1) and the dFdC-modified siRNAs Gem1,
Gem2, Gem5, Gem8 and Gem10, one can postulate stronger
interactions of the siRNA strands in the modified duplexes,
with the strongest interactions in the Gem5 (C5 - dFdC) and
Gem8 (C8 - dFdC) duplexes. By CD spectroscopy, we did
not observe any conformational changes for Gem1, Gem2,
Gem5, Gem8 and Gem10, in comparison to the NM duplex.
Therefore, in our opinion, the more positive entropy changes
for the NM duplex (DS1 = ꢁ164.92 cal Kꢁ1 molꢁ1) in
comparison to dFdC-modified siRNAs (DS1 in the range from
ꢁ172.15 cal Kꢁ1 molꢁ1 for Gem1 to ꢁ214.56 cal Kꢁ1 molꢁ1
for Gem5) reflect changes in the solvation entropy. Upon
strand binding, desolvation occurs, water is released and a
gain in solvent entropy is observed.
4. Experimental
4.1 Preparation of the phosphoramidite derivative of the dFdC
monomer and the synthesis of gemcitabine-modified RNA
oligonucleotides (Scheme 1)
3. Conclusions
In this paper, we have described the application of 20,20-
difluoro-20-deoxycytidine (dFdC, gemcitabine) modification
in the single substitution of a cytidine residue in the antisense
strand of the siRNA duplex. Encouraged by already published
results concerning fluorine-modified siRNAs and the unique
characteristics of gemcitabine,48 we decided to examine the
tolerance of such modifications at six sites of the antisense
strand crucial for siRNA silencing activity: in the ‘‘seed region’’
(positions 2–8) and at the cleavage site. The ‘‘seed region’’
is especially important for mRNA target recognition, and
complementarity to this sequence is often sufficient to obtain
the desired silencing. Detailed analysis has shown a position-
dependent tolerance for antisense strand modification.
50-Dimethoxytrityl-N4-benzoyl-20,20-difluoro-20-deoxycytidine
(1) (1 equiv., 0.4 mmol, 270 mg) was dissolved in anhydrous
acetonitrile (6 ml). A 0.5 M solution of ethylothiotetrazole
(1.6 equiv., 0.65 mM, 1.3 ml) in anhydrous acetonitrile and
2-cyanoethyl-bis-(N,N0-diisopropropyl)phosphoramidite (2)
(1.43 equiv., 0.57 mmol, 0.2 ml) were added, and the reaction
mixture stirred at room temperature for 1 h. The progress of
the reaction was monitored by TLC and 31P NMR. When
50-dimethoxytrityl-N4-benzoyl-20,20-difluoro-20-deoxycytidine
(1) was consumed (B2 h), the mixture was applied to a silica
gel column, and the product (3) eluted with 20% hexane in
CH2Cl2, followed by MeOH in CHCl3 (0–5% gradient). The
desired product was identified by 31P NMR (CDCl3): d 154.1,
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152.1, and FAB MS calculated for: C46H50O8N5P1F2 869.33,
found 870 [M + H]+, 868 [M ꢁ H]ꢁ. The synthesis of all
RNA oligonucleotides, unmodified and containing dFdC
units, was performed according to the phosphoramidite
approach42,43 on a Gene World DNA synthesizer (Department
of Bioorganic Chemistry, CMMS, PAS). The synthesis was
carried out on a 200 nmol scale using appropriately protected
phosphoramidite derivatives of thymidine, cytidine, uridine,
guanosine, adenosine and 20,20-difluoro-20-deoxycytidine (3),
LCA-CPG as a solid support and 5-benzylmercaptotetrazole
in anhydrous acetonitrile (0.25 M) as an activator. The
synthesis had a prolonged coupling time (up to 600 s) for
the modified unit. The coupling efficiency was determined by
the DMT-cation assay.
atmosphere of 5% CO2, the cells were washed with PBS and
lysed with NP-40 buffer (150 mM NaCl, 1% IGEPAL, 50 mM
Tris-HCl (pH 7.0), 1 mM PMSF) overnight at 37 1C. Cell
lysates were used for fluorescence determination.
4.4 Dual fluorescence assay
Fluorescence values of enhanced green fluorescent protein
(EGFP) and red fluorescent protein (RFP) fluorophores
were measured in cell-culturing plates using a Synergy HT
(BIO-TEK) reader. Data quantification was done with KC4
software. Excitation and emission wavelengths for GFP and
RFP were as follows: lex = 485/20 nm, lem = 528/20 nm for
GFP and lex = 530/25 nm, lem = 590/30 nm for RFP. The
siRNA activity was calculated as a ratio of GFP to RFP
fluorescence values. The level of GFP fluorescence of control
cells (transfected with pDsRed2-N1 and pBACE–GFP
plasmids, and control non-silencing siRNA) was taken as a
reference (100% GFP expression). Each siRNA activity value
given on the plots is an average of the mean value from three
independent experiments.
4.2 Deprotection and purification of oligonucleotides;
assembly of siRNAs
Oligonucleotides were cleaved from the solid support as
50-DMT-derivatives, and then deprotected and purified
according to a described procedure.49 Support-bound oligo-
nucleotides were treated with 33% ethanolic methylamine and
DMSO (Sigma-Aldrich) 1 : 1 (v : v) mixture for 15 min at
65 1C, then with triethylamine–trihydrofluoride (Sigma-
Aldrich) for 15 min at 65 1C. The reaction mixture was frozen
for 30 min at ꢁ20 1C, quenched with cold 1.5 M ammonium
bicarbonate and poured into a conditioned SepPak cartridge
(Waters). Shorter oligomers were eluted with 14% CH3CN in
50 mM NaOAc and 50 mM NaCl. The remaining oligomers
were treated with 2% aqueous TFA for 15 min at room
temperature and washed with water, 1 M NaCl, and water.
The product was eluted from the cartridge with 30% CH3CN.
The desired oligonucleotides were obtained in 90–95% yield.
The structure and purity of the oligomers were confirmed by
MALDI-TOF mass spectroscopy and 20% polyacrylamide/
7M urea gel electrophoresis. siRNA duplexes were assembled
in phosphate saline buffer (PBS, without Ca2+ and Mg2+) by
mixing equimolar amounts of complementary oligonucleo-
tides, heating at 95 1C for 2 min and slow cooling down to
room temperature (2 h). The formation of the resulting
duplexes was confirmed by 4% agarose electrophoresis.
4.5 Cytotoxicity determination
The cytotoxicity of siRNA duplexes for HeLa cells was
measured using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide assay (Sigma-Aldich) (activity
of the mitochondrial respiratory chain).50 Cells were plated
and transfected as described above (siRNAs at 1, 5, 10, 20 and
50 nM concentrations). As a background control, the cells
were treated with Lipofectamine 2000 only. After 48 h of
incubation at 37 1C and 5% CO2, 25 ml of MTT solution
(5 mg mlꢁ1 in PBS) was added to each well and incubated for
additional 2 h at 37 1C. Finally, 95 ml of lysis buffer (20% SDS,
50% aqueous dimethylformamide, pH 4.5) was added to each
well and incubated overnight at 37 1C. The absorbance of a
given sample was measured at 570 nm, with the reference
wavelength 630 nm (plate reader Synergy HT, BIO-TEK). The
percentage of living cells (PLC) was calculated from
the equation: PLC = ASpl ꢂ 100/(Acontrol cells), where ASpl is
the absorbance of a given sample of cells treated with siRNA
and Acontrol cells is the absorbance of the cells untreated with
siRNAs. Each data point represents the mean of three inde-
pendent measurements.
4.3 Cell cultures and transfections
HeLa cells were cultured in RPMI 1640 medium (Gibco,
Paisley) supplemented with 10% FBS (Gibco, BRL, Paisley)
and with antibiotics (penicillin 100 units mlꢁ1, streptomycin
100 mg mlꢁ1, Polfa) at 37 1C and 5% CO2. 24 h prior to the
experiment, cells were plated in 96-well black well plates with a
transparent bottom (Perkin-Elmer) at a density of 15 000 cells
per well. Before transfection, the cell medium was replaced
with the one free of antibiotics. The cells were cotransfected
4.6 Melting profiles and thermodynamic parameters
calculations
All UV absorption measurements were carried out in a 1 cm
path length cell with a UV/Vis 916 spectrophotometer
equipped with
a Peltier Thermocell (GBC, Australia).
Complementary oligonucleotides were mixed in 10 mM
Tris-HCl, 100 mM NaCl, 1 mM EDTA buffer (pH 7.4) at final
concentration of siRNA of 2 mM. The duplexes were heated at
96 1C and strand association was achieved by cooling down to
5 1C with a temperature gradient of 0.4 1C minꢁ1. Melting
profiles were recorded under heating from 5 to 96 1C with a
temperature gradient of 1 1C minꢁ1. The melting temperatures
were calculated by the first-order derivative method. Thermo-
dynamic parameters were obtained by numerical fitting of the
recorded curves (MeltWin software, version 3.5).
with DNA plasmids: pDsRed2-N1 (BD Biosciences), 15 ng wellꢁ1
,
pBACE-EGFP-C1,45 70 ng wellꢁ1 and siRNA (1 or 5 nM)
dissolved in Opti-MEM1 medium (Gibco, Paisley), and
complexed with the transfection reagent Lipofectamine 2000
(Invitrogen) according to the manufacturer’s protocol. The
cells were incubated in the transfection mixture for 5 h, and
then the mixture was replaced with fresh culturing medium
Containing antibiotics. After a 48 h incubation at 37 1C in an
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New J. Chem., 2010, 34, 918–924 | 923

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4.7 CD spectra recording
16 G. Meister, M. Landthaler, A. Patkaniowska, Y. Dorsett, G. Teng
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CD spectra in the range 200 to 350 nm were recorded on a CD
dichrograph (Jobin-Yvon) at 25 1C in the same buffer as in the
melting experiments at a duplex concentration of 2 mM, using
a 5 mm path length cell, 2 nm bandwidth and a 1–2 s
integration time. Each spectrum was smoothed with a
25-point algorithm (included in the manufacturer’s software,
version 2.2).
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The unmodified ssRNA sense strand was 50-labelled
with [g-32P] ATP by T4 polynucleotide kinase (Amersham)
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at 37 1C). Duplexes were formed by annealing equal molar
ratios of sense and antisense strands to form seven types of
siRNA (Fig. 2). Duplex formation was confirmed by 20%
PAGE under native conditions. Then duplexes (3 pmols per
reaction) were diluted in RPMI medium (Gibco) supple-
mented with 10% FBS (Gibco) and incubated at 37 1C.
Aliquots of 10 ml were collected after 0, 1, 5, 15, 30, 60, 120,
240, 360 min and 24 h, diluted in 1ꢂ loading buffer (Fermentas),
frozen in liquid N2 and kept at ꢁ20 1C until analysis time.
Control sample ‘‘0’’ was prepared with ice cold FBS and
RPMI medium, collected and immediately frozen. Samples
were separated on 20% PAGE under non-denaturing
conditions. Substrate and degraded products were quantified
using a G-Box (SynGene, Cambridge, UK) instrument and
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Acknowledgements
We thank Professor Stec for scientific inspiration, and
Professor Barbara Nawrot for her assistance and scientific
support of the presented studies. This work was supported by
PBZ-MNiSW-07/I/2007 for years 2008–2010 and by the
Bioorganic Chemistry and Structural Biology Network.
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This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2010
924 | New J. Chem., 2010, 34, 918–924