Synthesis, physico-chemical and biological properties of DNA and RNA oligonucleotides containing short alkylamino internucleotide bond

Article abstract of DOI:10.1135/cccc2011090

The condensation of the 5'-O-DMT-3'-deoxy-3'-aminothymidine with 3'-O-TBDMS-thymidine- 5'-aldehyde, followed by reduction of the resultant imine derivative and removal of tertbutyldimethylsilyl (TBDMS) protecting group, provided a dimer (denoted as T

Full text of DOI:10.1135/cccc2011090

Oligonucleotides with Modified Backbone
1471
SYNTHESIS, PHYSICO-CHEMICAL AND BIOLOGICAL PROPERTIES OF
DNA AND RNA OLIGONUCLEOTIDES CONTAINING SHORT
ALKYLAMINO INTERNUCLEOTIDE BOND
b
a2
Milena SOBCZAKa1, Katarzyna KUBIAK , Magdalena JANICKA
,
Malgorzata SIERANTa3, Barbara MIKOLAJCZYK and Barbara NAWROT
*
a4
a5,
a
Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies
of Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland;
e-mail: milena@bio.cbmm.lodz.pl, mjanicka@bio.cbmm.lodz.pl, msierant@bio.cbmm.lodz.pl,
barbaram@bio.cbmm.lodz.pl, bnawrot@bio.cbmm.lodz.pl
Institute of Technical Biochemistry, Technical University of Lodz,
1
2
3
4
5
b
Stefanowskiego 4/10, 90-924 Lodz, Poland; e-mail: katarzyna.kubiak@p.lodz.pl
Received April 28, 2011
Accepted July 14, 2011
Published online December 7, 2011
This paper is dedicated to Professor Antonín Holý, on the occasion of his 75th birthday.
The condensation of the 5′-O-DMT-3′-deoxy-3′-aminothymidine with 3′-O-TBDMS-thymidine-
5′-aldehyde, followed by reduction of the resultant imine derivative and removal of tert-
butyldimethylsilyl (TBDMS) protecting group, provided a dimer (denoted as TNHT), which
is a congener of dithymidine phosphate with the phosphate linkage 3′-O-P(O)(OH)-O-5′
replaced with an amino group (–NH–). After phosphitylation of the 3′-OH group, the dimer
T
NHT was introduced (by the standard phosphoramidite approach) into a central part of the
nonadecathymidylate. This oligomer exhibited lower affinity to the complementary single
and double stranded DNA complements as compared to unmodified T19 oligonucleotide.
The cleavage of modified oligomer with the snake venom and calf spleen
phosphodiesterases was completely suppressed at the site of modification. RNA oligomers
containing the TNHT dimer were used for preparation of siRNA molecules directed towards
mRNA of BACE1 (beta-site amyloid precursor protein cleaving enzyme). The presence of the
T
NHT units at the 3′-ends of the RNA strands of the siRNA molecule (the siRNA itself is an
effective gene expression inhibitor for BACE1) preserved the gene silencing activity and im-
proved the stability of the modified siRNA in 10% fetal bovine serum.
Keywords: Schiff base; Oligonucleotide; Bioorganic chemistry; Modified oligonucleotide;
Modified backbone; Thymidine dimer.
Oligonucleotides are useful tools for molecular biology, biotechnology and
medicinal chemistry. In the latter application, oligonucleotides are used as
sequence-specific inhibitors of gene expression at the transcription level
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 12, pp. 1471–1486
© 2011 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2011090

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Sobczak, Kubiak, Janicka, Sierant, Mikolajczyk, Nawrot:
(antigene, decoy and CpG oligonucleotides) as well as at the post-
transcriptional level (antisense oligonucleotides, ribozymes and short inter-
fering RNAs). One of the most discouraging features of the therapeutic
oligonucleotides is their fast degradation by cellular nucleases. This dis-
advantage is less severe when more nucleases resistant oligonucleotides
containing chemically modified internucleotide linkages are used. Oligo-
nucleotide phosphorothioates were the first chemically modified constructs
with enhanced resistance in body fluids. Because of the polyanionic charac-
ter of oligonucleotides, some carriers (e.g. lipids) are used to facilitate their
cellular up-take. Well known replacement of the phosphate internucleotide
bond with methylphosphonate function gives rise to the nuclease resistant,
non-charged oligomers with improved ability to cross the cell membrane.
However, both phosphorothioate and methylphosphonate analogs are
P-chiral, and the corresponding oligomers, if prepared on non-stereo-
controlled way, exist as mixtures of hundreds or thousands P-diastereo-
meric species1. Thus, nucleases resistant oligonucleotides with achiral and
non-ionic internucleotide linkages can be considered as a next generation
of antisense oligonucleotides2. Oligodeoxyribonucleotides having the phos-
phate group replaced with carbonate3, carbomethoxyl4, carbamate or oxy-
amide function5, were first constructs of this type, followed by those with
alkylamino-6 or amido-type7 internucleotide linkages. Recently, it was dem-
onstrated that amide residues are excellent mimics of the phosphate link-
ages in the RNA backbone8. Heterocyclic internucleotide linkages, based on
imidazole6e or triazole rings, constitute another type of phosphate mimics9.
The latter were successfully synthesized by the click chemistry ([3+2]
azide-alkyne cycloaddition). In this paper we describe the chemical syn-
thesis and physico-chemical and biological properties of DNA and RNA
oligonucleotides containing a novel unit, in which the internucleotide
phosphate bond 3′-O-P(O)(OH)-O-5′ is replaced with an amino function
(–NH–).
RESULTS AND DISCUSSION
Chemical Synthesis of a Dimer TNH
T
Condensation of 5′-O-DMT-3′-amino-3′-deoxythymidine10 1 (Scheme 1)
with 3′-tert-butyldimethylsilylthymidine-5′-aldehyde11 2 (the latter ob-
tained from 3′-tert-butyldimethylsilylthymidine by Swern’s oxidation12)
resulted in the formation of a Schiff base 3. Reduction of the imine group
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Oligonucleotides with Modified Backbone
1473
in 3 with NaBH(OAc)3 and chromatographic purification provided the
dimer 4. It was characterized by FAB MS (in a negative ions mode) and the
expected signal m/z 880 (m.w. 881) was found. Removal of the 3′-protecting
group with TBAF in THF 13 provided the dimer 5, which after routine purifi-
cation (a silica gel column) was phosphitylated with O-(2-cyanoethyl)-
N,N-diisopropylchlorophosphoramidite14. The crude 6 was purified by
chromatography on a silica gel column and lyophilized from benzene. It
was further used for the synthesis of the model oligonucleotides via the
phosphoramidite method (Table I).
Chemical Synthesis of Oligonucleotides 7–9
Synthesis of oligonucleotides was performed according to the manufac-
turer’s recommendations, except for the coupling time of the modified
unit, which was extended to 600 s. An oligodeoxyribonucleotide T9TNHTT8
was synthesized on the LCA-CPG solid support, then routinely deprotected
and purified by the two step procedure15. The 5′-O-DMT-protected
(DMT-on) and fully deprotected (DMT-off) oligomer 7 was subjected to
SCHEME 1
Chemical synthesis of 3′-O-phosphoramidite derivative of 5′-O-DMT-protected dimer TNHT
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Sobczak, Kubiak, Janicka, Sierant, Mikolajczyk, Nawrot:
RP-HPLC on an ODS Hypersil C18 column. The structure of this oligomer
was confirmed by matrix-assisted laser desorption/ionization time-of-flight
(MALDI-TOF) mass spectrometry at both steps (Table I).
TABLE I
The sequences of prepared oligonucleotides containing the TNHT dimer and their MALDI-TOF
mass spectral analysis data
MALDI-TOF
MS, m/z
ODN No.
Sequence
M.w.
7 (DNA)
5′-T9TNHTT8-3′
5937
5944
(DMT-on)
5634
(DMT-on)
5639
(DMT-off)
(DMT-off)
8 (siRNA sense strand)
5′-AAUCAGACAAGUUCUUCAUTNHT-3′
6519
6576
6515
6574
9 (siRNA antisense strand) 3′-TNHTUUAGUCUGUUCAAGAAGUA-5′
The RNA oligonucleotides 8 and 9, corresponding to the sense and
antisense strands of siRNA directed towards beta-site amyloid precursor pro-
tein cleaving enzyme (BACE1) mRNA 16, were synthesized on a universal
solid support17 (Glen Research) and cleaved from the support according to
the manufacturer’s protocol (NH3 in MeOH, 30 min, room temperature).
After removal of the 2′-protecting group with Et3N·3HF, the resulting prod-
uct was purified by RP-HPLC (a PRP-1 column, Hamilton). The structures of
8 and 9 were confirmed by MALDI-TOF mass spectrometry (Table I), and
their purity was assessed by electrophoresis in 20% polyacrylamide/7 M
urea gel (data not shown).
Thermal Stability of the Complexes of T9TNHTT8 with
dA19 and d(A21C4T21) Templates
Thermal stability of the complexes of fully deprotected oligonucleotide 7
with the complementary single stranded dA19 and double stranded
d(A21C4T21) oligodeoxyribonucleotides was determined to assess the affinity
of the modified oligonucleotide towards target molecules. This parameter is
important when antisense/antigene properties are considered. The dA21
tract of the d(A21C4T21) hairpin oligonucleotide is able to bind 7 (as the
third strand) by the Hoogsteen hydrogen bonds. The melting temperature
measurements were performed in Tris-HCl buffer, pH 7.4. Since a typical
pKa of dialkyl amines is above 10 (for diethyl amine pKa = 10.87 18) one
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Oligonucleotides with Modified Backbone
1475
may assume that the –NH– linkage in the TNHT dimer exists in a protonated
form. We found that the TNHT modification present in the central part of
the duplex 7/dA19 decreases its melting temperature by 4.8 °C, compared to
the Tm of the reference T19/dA19 duplex (Tm = 48.1 vs 52.9 °C), while the
triplex 7/d(A21C4T21) melts at the temperature only slightly lower than its
non-modified counterpart (Tm = 31.0 vs 32.6 °C). These data indicate that
the inserted TNHT dimer (in which there is only a two bonds linkage be-
tween the C3′ atom of the upstream T unit and the C5′ atom of the down-
stream T unit) decreases the hybridization affinities of the modified strand.
It is rather expected feature, although there are exceptions, like 5′-nor-
oligonucleotides with a shortened sugar-phosphate backbone (due to the
lack of the 5′-methylene group), which form only slightly less stable com-
plexes with DNA templates (∆Tm = 9 °C between d(5′-nor-A)24/T24 and
dA24/T24, less than 0.4 °C/modification)19. In a case of the triplex 7/
d(A21C4T21), the destabilizing effect of the shortening (∆Tm = 1.6 °C) is not
very big. Perhaps, to a certain extent it is compensated by electrostatic at-
traction between the protonated NH function and the negatively charged
phosphate groups in the template.
Circular Dichroism Spectra of Complexes (Duplex and Triplex)
Containing T9TNHTT8 strand
The overall structures of the complexes of oligonucleotide 7 with dA19 and
d(A21C4T21) were analyzed by circular dichroism (CD) spectroscopy and
compared to the structures of the parent non-modified complexes. The
spectra for complexes with the strands stoichiometry 1:1, recorded in a
range of 220–320 nm at 25 °C, are shown in Fig. 1. The CD spectra for
duplexes 7/dA19 and T19/dA19 are similar and have a profile typical for the
B-type DNA helix (Fig. 1a). The CD spectra for triplexes 7/d(A21C4T21) and
T19/d(A21C4T21) differ slightly in a range of 220–230 nm (an increase of in-
tensity) as well as in 245–263 nm (a decrease of intensity for the modified
triplex; Fig. 1b), indicating that the modified unit disturbs the helical struc-
ture of the B-type DNA 20
.
Nucleolytic Stability of Oligonucleotides Containing TNHT Moieties
A) Stability of 7 in the presence of snake venom and calf spleen phosphodi-
esterases. Oligonucleotide 7 was treated with snake venom phosphodi-
esterase (svPDE, PDE I, 3′-exonuclease) and with calf spleen phospho-
diesterase (csPDE, PDE II, 5′-exonuclease). The reaction was stopped at
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1476
Sobczak, Kubiak, Janicka, Sierant, Mikolajczyk, Nawrot:
given time points and the products of digestion were analyzed by
MALDI-TOF mass spectrometry. Figures 2a and 2b show the series of four
consecutive MS spectra for digestion products, recorded after 10, 30, 45 and
120 min of incubation of 7 with the csPDE and svPDE nucleases, respec-
4.5
a
3.5
2.5
1.5
0.5
–0.5
–1.5
–2.5
–3.5
–4.5
–5.5
210
230
250
270
290
310
Wavelength, nm
2.5
1.5
b
0.5
–0.5
–1.5
–2.5
–3.5
–4.5
–5.5
210
230
250
270
290
310
Wavelength, nm
FIG. 1
CD spectra of a duplexes T19/dA19 (᭺) and 7/dA19 (ꢀ) and b triplexes T19/d(A21C4T21) (᭺) and
7/d(A21C4T21) (m). Equimolar amounts of modified oligonucleotides and complementary
templates (c = 1 µM of each strand) were dissolved in 10 mM Tris-HCl buffer, pH 7.5, containing
10 mM MgCl2 and 100 mM NaCl; the spectra were recorded in a range of 220–320 nm at 25 °C
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Oligonucleotides with Modified Backbone
1477
tively. The substrate 7 (m.w. 5634, m/z 5634) is cleaved to yield shorter
oligonucleotides, which differ by 304 atomic mass units (u), until the en-
zyme reaches the modification site. Upon digestion with csPDE and svPDE,
the nucleolytically stable products TNHTT8 (m/z 2895) (Fig. 2a) and T9TNH
T
(m/z 3199) (Fig. 2b), respectively, were found. This means that, as it was ex-
pected, the modified internucleotide bond (structurally far different from
a
b
FIG. 2
MALDI-TOF mass spectra of products of the digestion of 7 with calf spleen phosphodiesterase
(a) and snake venom phosphodiesterase (b). The samples were digested for 10, 30, 45 and 120
min at 25 °C
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1478
Sobczak, Kubiak, Janicka, Sierant, Mikolajczyk, Nawrot:
the phosphate moiety) remained undigested and prevented the partially
hydrolyzed products from further nucleolytic cleavage.
B) Stability of siRNA containing TNHT dimer at the 3′-ends in 10% fetal bovine
serum. Short interfering RNAs (siRNAs) are executory molecules of the RNA
interference, which was discovered recently as a gene expression regulatory
mechanism. siRNAs are commonly used as exogenous gene silencers. They
consist of complementary RNA strands, which contain two non-paired
units at their 3′-ends. It was already shown that siRNAs with deoxyribo-
nucleotide TPT overhangs are as active as their full-RNA congeners. To ex-
amine the influence of the TNHT modification on the silencing activity and
nucleolytic stability of siRNA, we tested duplexes modified at the 3′-ends in
one or both strands. Three siRNA duplexes designed for silencing of BACE1
protein16,21 were prepared by annealing the sense (8 or 10) and antisense
(9 or 11) strands (Table II), and their stability in 10% fetal bovine serum
(FBS) was quantified.
TABLE II
The sequences of siRNA duplexes used in these studies. Non-modified parent single stranded
RNAs are designed as sense (10) and antisense (11) strands
siRNA
Sequence
S1 (10/11)
5′-AAUCAGACAAGUUCUUCAUTT-3′
3′-TTUUAGUCUGUUCAAGAAGUA-5′
5′-AAUCAGACAAGUUCUUCAUTNHT-3′
3′-TNHTUUAGUCUGUUCAAGAAGUA-5′
S2 (8/9)
S3 (10/9)
5′-AAUCAGACAAGUUCUUCAUTT-3′
3′-TNHTUUAGUCUGUUCAAGAAGUA-5′
SiRNAs S1, S2 and S3 as well as single stranded oligonucleotides 8, 9, 10
and 11 were treated with 10% FBS in RPMI medium at 37 °C for up to 24 h.
At consecutive time points, aliquots were removed and examined for the
intact single stranded RNA or the duplex by native polyacrylamide gel elec-
trophoresis (PAGE; Fig. 3).
Despite of the presence of the TNHT modification in the single stranded
RNAs 8 and 9, these oligomers were degraded as efficiently as non-modified
RNAs 10 and 11. Double stranded RNAs were more resistant to nucleolytic
degradation. There was an initial loss of the duplexes – in each case the dif-
ference in amount of the intact duplex was observed between a sample
without FBS and a sample “0” with cold FBS, incubated in ice. Remaining
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Oligonucleotides with Modified Backbone
1479
amounts of duplexes were degraded slowly, but no significant difference in
stability was noticed between the wild type and modified siRNAs. The half
life times for all duplexes were around 5 h. These results indicate that the
T
NHT modification present at the 3′-ends of siRNAs increases their stability
toward FBS to a little extent only.
a
0
5
10 15 20 30 60 120 240 360 480 1440 min
FBS(+)
FBS(–)
siRNA S1
siRNA S2
siRNA S3
b
120
siRNA S1
siRNA S2
siRNA S3
100
80
60
40
20
0
0
6
12
18
24
Time, h
FIG. 3
Stability of siRNA duplexes in 10% FBS. a Double stranded RNA (siRNAs S1, S2 and S3) were
treated with 10% FBS at 37 °C for indicated times 0, 5, 10, 15, 20, 30, 60, 120, 240, 360,
480 min and 24 h, then were examined by non-denaturating gel electrophoresis to determine
the amount of intact duplexes. b The bands were quantified by ImageQuant v. 5.0 software
(Molecular Dynamics) and the percentage of the intact siRNA (compared to the initial
amount) was calculated. Data represent mean values from two independent experiments
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Sobczak, Kubiak, Janicka, Sierant, Mikolajczyk, Nawrot:
Gene Silencing Activity of siRNA S2 Containing TNHT Units
at 3′-End of Each Strand
Gene silencing activity of S2 was determined in a dual fluorescence assay,
conducted in HeLa cells16,21, and was compared to the activity of the par-
ent, non-modified S1 and siRNA “B3”. The latter duplex was already de-
scribed in the literature as an active BACE1 silencer22. Cells treated with
lipofectamine only were used as a control (C). The duplexes were tested at
1 nM and 100 pM concentrations. The results (presented in Fig. 4) are mean
values from three independent transfection experiments.
These results indicate that at 1 nM concentration the duplex containing
the TNHT dimer at the 3′-ends of both strands induces gene silencing almost
as efficiently as the non-modified counterpart (~5 vs 2% of BACE-GFP
expression). The duplex is still active, but to a smaller extent (40 vs 15% of
BACE-GFP expression) at ten-fold lower concentration (100 pM).
In conclusion, in this paper we have investigated a new type of the
internucleotide bond, in which the phosphate moiety 3′-O-P(O)(OH)-O-5′
is replaced with an amino group –NH–. The thymidine dimer (TNHT) con-
taining such a short bond, which most likely exists in the protonated form,
was introduced into DNA and RNA oligomers by standard phosphoramidite
approach. DNA oligomer T9TNHTT8 exhibited lower affinity to the comple-
120
100 100
100
74
80
60
40
40
22
15
20
5
2
siRNA S1
0
C
B3
siRNA S2
FIG. 4
Gene silencing activity of siRNA S1, siRNA S2 and siRNA “B3” used in 1.0 (᭿) and 0.1 nM (ᮀ)
concentration determined by a dual fluorescence assay in HeLa cells; control cells treated with
lipofectamine only (C)
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Oligonucleotides with Modified Backbone
1481
mentary single and double stranded DNA complements as compared to T19
parent oligonucleotide. Its hydrolysis with snake venom and calf spleen
phosphodiesterases was completely suppressed at the site of modification.
RNA oligomers containing TNHT dimer at the 3′-ends were used for prepara-
tion of siRNA molecules, directed towards BACE1 mRNA. Such TNHT-
modified siRNAs exhibited similar silencing activity as non-modified siRNA
duplex, while their stability in 10% fetal bovine serum was slightly en-
hanced. Therefore, oligonucleotides with this nuclease stable linkage can be
considered as candidates for biochemical probing or therapeutic use.
EXPERIMENTAL
The nuclear magnetic resonance spectra were recorded with a Bruker AC-200 instrument
at 200.13 MHz for 1H and 81.33 MHz for 31P. Chemical shifts (δ-scale) are given in ppm,
coupling constants (J) in Hz. Unless otherwise indicated, the samples were dissolved in
CDCl3. The solvent signal was used as the internal standard for 1H (δ CDCl3 = 7.26 ppm)
and 85% H3PO4 as the external standard for 31P. Fast atom bombardment (FAB) mass spectra
were recorded on a Finnigan MAT 95 spectrometer and MALDI-TOF mass spectra were re-
corded in positive and negative ion modes on a Voyager–Elite instrument (Perseptive
Biosystem, Inc., Framingham (MA), USA). Unmodified oligomer T19 was prepared by the
phosphoramidite method on an ABI 394 synthesizer. Preparative purifications and separa-
tions were performed on a ThermoQuest Hypersil ODS 5 µ column (250 × 4.6 mm) using a
binary analytical HPLC system and a UV/VIS-151 detector (Gilson). Aqueous triethylamine
bicarbonate (0.1 M, pH 7.5)/acetonitrile buffer system was used as an eluent at a flow rate
1 ml/min. Evaporations were carried out at 40 °C. TLC was carried out on silica gel 60F254
plates (Merck, Germany) in chloroform/methanol (95:5 (A) or 90:10 (B)) solvent system.
Nucleosides were purchased from Pharma Waldhof (Germany). Chloroform used for chro-
matography always contained triethylamine (1%, v/v).
Synthesis of 3′-O-tert-Butyldimethylsilylthymidine-5′-aldehyde (1)
Thymidine derivative 1 was obtained according to the already published procedure12
.
Briefly, 0.2 ml of oxalyl chloride (1.65 mmol, 3 eq.) was dissolved in freshly distilled CH2Cl2
(10 ml), dried over CaH2, and after 15 min the solution was cooled down to –78 °C. Then,
0.25 ml of dimethylsulfoxide (DMSO; 3.3 mmol, 6 eq.) was added, the reaction mixture was
stirred for 10 min, and 200 mg of 3′-O-tert-butyldimethylsilylthymidine13 (0.55 mmol, 1 eq.,
dried overnight at vacuum over P2O5) dissolved in 2 ml of anhydrous CH2Cl2 was added
dropwise over ca. 5 min. The mixture was stirred for 30 min and 0.4 ml of triethylamine
(2.75 mmol, 5 eq.) were added. After next 30 min the reaction mixture was warmed up to
the room temperature, diluted with CH2Cl2 (20 ml) and washed with 1% aqueous HCl
(10 ml) and 5% aqueous NaHCO3 (10 ml). The organic layer was dried with MgSO4 and the
solvent was evaporated to yield the aldehyde 1 (174 mg, 87%). RF 0.53 (A). 1H NMR (CDCl3,
200 MHz): 9.49 (1 H, s, H-C(O)), 7.40 (1 H, d, JCH3-H6 = 1.2, H-6), 6.22–6.19 (1 H, m, H-1′),
4.51–4.47 (1 H, m, H-3′), 3.97–3.71 (2 H, m, H-4′, H-5′), 2.47–2.18 (2 H, m, H-2′, H-2”), 1.90
(3 H, d, JCH3-H6 = 1.2, CH3 Thy), 0.89 (9 H, s, t-Bu), 0.08 (6 H, s, Si-CH3).
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Sobczak, Kubiak, Janicka, Sierant, Mikolajczyk, Nawrot:
Condensation of Thymidine-5′-aldehyde 1 with 5′-O-(4,4′-Dimethoxytrityl)-
3′-amino-3′-deoxythymidine 2
To the solution of 106 mg of thymidine derivative 1 (0.38 mmol, dried in vacuum for 2 h)
in 8 ml of tetrahydrofurane (THF), anhydrous MgSO4 (ca. 100 mg) and 5′-O-DMT-3′-amino-
3′-deoxythymidine 2 (207 mg, 0.38 mmol) were added. The reaction mixture was stirred at
room temperature overnight, filtered and concentrated to dryness at reduced pressure. The
residue was applied on a silica gel column and chromatographed with a gradient of metha-
nol in chloroform (0 to 5% MeOH). The dimer 3 was obtained in 68% yield (246 mg).
RF 0.52 (A). 1H NMR (CDCl3, 200 MHz): 7.73 (1 H, m, H-5′, 3′-T), 7.69 (1 H, d, JCH3-H6 = 1.0,
H-6), 6.93 (1 H, d, JCH3-H6 = 1.0, H-6), 7.36–6.80 (13 H, m, aromatic DMT), 6.82 (1 H, m,
H-1′, 3′-T), 6.31 (1 H, m, H-1′, 5′-T), 5.58 (1 H, m, H-3′, 3′-T), 5.30 (1 H, m, H-4′, 3′-T), 4.17
(1 H, m, H-3′, 5′-T), 4.17 (1 H, m, H-4′, 5′-T), 3.78 (6 H, s, 2 × CH3 of DMT), 3.63 and 3.10
(2 H, 2 × m, H-5′ and H-5”, 5′-T), 3.35 and 2.76 (2 H, 2 × m, H-2′ and H-2”, 3′-T), 2.62 and
2.42 (1 H, m, H-2′ and H-2”, 5′-T), 1.91 (3 H, d, JCH3-H6 = 1.0, CH3 Thy), 1.53 (3 H, d,
JCH3-H6 = 1.0, CH3 Thy), 0.87 (9 H, s, t-Bu), 0.07 (6 H, s, 2 × CH3(Si)). FAB MS: 878.7 [M –
H]–, m.w. for C47H57N5O10Si 879.
Reduction of the Thymidine Dimer 3
Thymidine dimer 3 (240 mg, 0.3 mmol) was dissolved in anhydrous ethanol (10 ml) and
treated with NaBH(OAc)3 (130 mg, 0.6 mmol, 2 eq.). After 2 h of stirring at room tempera-
ture, the mixture was concentrated under reduced pressure and the crude reaction product
was chromatographed on a silica gel column with a gradient of methanol in chloroform in
(0 to 5% MeOH). The dimer 4 was obtained in 88% yield (211 mg). RF 0.50 (A). FAB MS:
880.4 [M – H]–, m.w. for C47H59N5O10Si 881.
Removal of the tert-Butyldimethysilyl Group from 4
Thymidine dimer 4 (88 mg, 0.1 mmol) was dried in vacuum overnight, dissolved in anhy-
drous THF (1 ml) and treated with 1 M solution of tetra-(n-butylammonium) fluoride (TBAF)
in THF (0.45 mmol, 0.45 ml). After 2 h of stirring at room temperature, the mixture was di-
luted with chloroform (20 ml) and washed with water (10 ml). The organic layer was dried
with MgSO4, the solvent was evaporated and the crude product was chromatographed on a
silica gel column with a gradient of methanol in chloroform (0 to 7% MeOH). The product
5 was obtained in 94% yield (173 mg). RF 0.31 (A). 1H NMR (CDCl3, 200 MHz): 7.61 (1 H, d,
JCH3-H6 = 1.0, H-6), 7.24 (1 H, d, JCH3-H6 = 1.0, H-6), 7.36–6.80 (13 H, m, aromatic DMT),
6.29 (1 H, m, H-1′, 5′-T), 6.11 (1 H, m, H-1′, 3′-T), 4.43 (1 H, m, H-3′, 3′-T), 3.80 (1 H, m,
H-4′, 3′-T), 3.80 (1 H, m, H-3′, 5′-T), 3.78 (6 H, s, 2 × CH3 of DMT), 3.55 (1 H, m, H-5′, 5′-T),
3.45 (1 H, m, H-5”, 5′-T), 3.32 (1 H, m, H-4′, 5′-T), 2.89 (2 H, m, H-5′ and H-5”, 3′-T),
2.35–2.21 (2 H, m, H-2′ and H-2”, 3′-T), 2.35–2.21 (2 H, m, H-2′ and H-2”, 5′-T), 1.83 (3 H,
d, JCH3-H6 = 1.0, CH3 Thy), 1.47 (3 H, d, JCH3-H6 = 1.0, CH3 Thy). FAB MS: 768.3 [M + H]+
and 766.3 [M – H]–, m.w. for C41H45N5O10 767.
Synthesis of the Monomer 6
To the solution of thymidine dimer 5 (76 mg, 0.01 mmol) and N,N-diisopropylethylamine
(60 µl, 0.03 mmol, 3 eq.) in anhydrous acetonitrile (2 ml) O-(2-cyanoethyl)-N,N-diiso-
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Oligonucleotides with Modified Backbone
1483
propylchlorophosphoramidite (25 µl, 0.015 mmol, 1.5 eq.) was added dropwise using a
gas-tight syringe. After 30 min of stirring, the TLC analysis showed the presence of the sub-
strate 5, therefore, the next portion of the phosphitylating reagent (10 µl) was added
dropwise and the reaction mixture was stirred for additional 30 min. Subsequently, the reac-
tion mixture was applied onto a silica gel column and the product was chromatographed
with a gradient addition of methanol in ethyl acetate/hexane (1:1, v/v) (0 to 7% MeOH).
The product 6 was isolated in 67% yield (34 mg). RF 0.36 (B). 31P NMR (CDCl3, 200 MHz):
148.98 and 148.78.
Synthesis and Purification of Oligonucleotide T9TNHTT8
The synthesis of oligomer T9TNHTT8 (7) was performed at 1-µmol scale on an ABI 394 syn-
thesizer (Applied Biosystems Inc., Foster City (CA), USA) using commercially available
5′-O-DMT-thymidine-3′-O-(O-2-cyanoethyl-N,N-diisopropyl)phosphoramidite (Glen Research)
and the monomer 6. A succinyl-linked LCA-CPG was used as a solid support. The only
change in the manufacturer recommended protocol of the synthesis was a prolonged cou-
pling time (600 s) for the modified monomer 6. The coupling efficiency of 6 determined by
DMT-ion assay was 92%. The resultant oligomer carrying the 5′-terminal DMT group15 was
cleaved off from the solid support by treatment with 30% ammonium hydroxide (1 ml) at
55 °C for 16 h and purified by a standard RP-HPLC. The 5′-DMT group was removed by
treatment with 50% acetic acid at room temperature for 30 min, followed by RP HPLC puri-
fication on a PRP-1 Hamilton column (305 × 7 mm). Fully deprotected oligonucleotide was
obtained in 76% yield. The structure and purity of the oligomer were confirmed by
MALDI-TOF mass spectrometry, 20% polyacrylamide/7 M urea gel electrophoresis (PAGE),
and RP HPLC analysis. Spectral and chromatographic characteristics of the oligomer
T9TNHTT8 (7) are given in Table I.
Synthesis and Purification of RNA Oligonucleotides 8 and 9
RNA oligonucleotides were synthesized on a 1-µmol scale, using an Applied Biosystems 394
instrument under conditions recommended by the manufacturer. The synthesis was per-
formed on the Universal solid support II (Glen Research)17. The first coupling performed
with the thymidine dimer 6 was followed by commercial RNA phosphoramidite monomers
(Glen Research). The oligomers (the DMT protecting group was removed at the end of syn-
thesis) were deprotected within the nucleobases and cleaved from the solid support by treat-
ment with NH3 in MeOH at room temperature for 30 min. The solutions were decanted and
the solvent was evaporated under reduced pressure (Speed Vac). The resulting products were
treated with 250 µl of hydrogen fluoride–triethylamine complex (Aldrich) at room tempera-
ture for 24 h and then with 1 ml of n-butanol. The reaction mixtures were kept in a freezer
at –20 °C for 30 min. The pellets were spun down and washed once with diethyl ether. The
crude products were dissolved in deionized water and isolated by means of RP-HPLC
(a PRP-1 Hamilton column, 305 × 7 mm). The structure of oligomers was confirmed
by MALDI-TOF mass spectrometry and their purity was assessed by electrophoresis in
20% polyacrylamide/7 M urea gel. Spectral and chromatographic characteristics of oligomers
8 and 9 are given in Table I.
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1484
Sobczak, Kubiak, Janicka, Sierant, Mikolajczyk, Nawrot:
Melting Temperature Measurements
UV thermal stability studies of oligonucleotides (Table I) were performed on a Cintra 40
UV-Vis spectrophotometer (GBC, Australia). The duplexes consisting of the modified oligo-
nucleotide and the complementary template (Table I) were dissolved in 10 mM Tris-HCl
buffer, pH 7.4, containing 100 mM NaCl and 5 mM MgCl2, whereas the heteroduplexes of
the modified oligonucleotide and a single DNA target (Table II) were dissolved in an appro-
priate buffer. For proper annealing, the samples were heated to 70 °C and cooled to 5 °C at
a rate of 0.5 °C/min. The samples were kept for 5 min at 5 °C and heated to 86 °C at a rate
of 0.2 °C/min. Melting profiles for the heteroduplexes were recorded for equimolar amounts
of modified oligonucleotides and complementary template (1 µM of each strand) at 260 nm.
The melting temperatures obtained from the melting curves using the first order derivative
method allowed for calculation of thermodynamic parameters (data not shown).
CD Spectra Recording
CD spectra were recorded on a CD6 dichrograph (Jobin–Yvon, Longjumeau, France) using a
cell with 0.5-cm path length. The bandwidth of 2 nm and 1–2 s integration time were ap-
plied. Each spectrum was smoothed with a 25-point algorithm (included in the manufac-
turer software, version 2.2.1). The spectra for duplexes (1.0 µM each strand) of modified
oligonucleotide with complementary target strand (Table II) in appropriate restriction buffer
were recorded in a range of 200–320 nm at 25 °C.
Assay for digestion of oligonucleotide T9TNHTT8 with snake venom phosphodiesterase
(svPDE) or calf spleen phosphodiesterase (csPDE) and MALDI-TOF analysis23
.
Samples of oligonucleotide T9TNHTT8 (0.1 A260) were mixed with svPDE (0.1 mU, 5 µl
buffer solution 2.5 mM Tris HCl, pH 8.5, 0.5 mM MgCl2) or with csPDE (0.4 mU, 5 µl acetate
buffer (50 mM, pH 5.0)) and incubated at 37 °C. After 10, 30, 45 and 240 min, 1 µl aliquots
were withdrawn, mixed with 1 µl of the matrix (2,4,6-trihydroxyacetophenone (10 mg/ml in
water/acetonitrile, 1:1) – ammonium citrate dibasic (50 mg/ml in water), 8:1, v/v) and
applied directly to the sample plate. After 10 min of drying, the samples were analyzed by
MALDI-TOF mass spectrometry using a Voyager Elite instrument (PerSeptive Biosystems, USA).
siRNA Stability in 10% FBS
Unmodified ssRNA (3′ TPT) and modified ssRNA (3′ TNHT) strands were labeled at the 5′-OH
group with [γ-32P]ATP by T4 polynucleotide kinase (Amersham) (1 h, 37 °C). Duplexes were
formed by annealing of equimolar amounts of unmodified or modified sense and antisense
strands to form three types of siRNA: S1 (10/11), S2 (8/9), S3 (10/9) (Table II). Duplex
formation was confirmed by 20% PAGE under native conditions. Then duplexes (3 pmol/
reaction) were diluted with the RPMI medium (Gibco) supplemented with 10% FBS (Gibco)
and incubated at 37 °C. Aliquots of 10 µl were collected after 0, 5, 10, 15, 20, 30, 60, 120,
240, 360, 480 min and 24 h, diluted in 1× loading buffer (Fermentas), frozen in liquid nitro-
gen and kept at –20 °C until analyzed. Control sample “0” was prepared in ice in RPMI me-
dium and collected immediately after mixing with cold fetal bovine serum (FBS). The
samples were separated in 20% PAGE under non-denaturing conditions. Gels were analyzed
with PhosphoImager (Molecular Dynamics) and quantified by ImageQuant v. 5.0 software.
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Oligonucleotides with Modified Backbone
1485
siRNA Activity Determined in a Dual Fluorescence Assay
HeLa (human cervical carcinoma) cells were cultured in RPMI 1640 medium (Gibco, BRL,
Paisley), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, BRL, Pais-
ley) and 100 U/ml penicillin and 100 µg/ml streptomycin (Polfa) at 37 °C and 5% CO2. One
day before transfection, the cells were trypsinized, diluted with fresh media and plated in
black-wall 96-well plates (with a transparent bottom, Perkin–Elmer) at a density of 1.5 × 104
cells per well. Before transfection, the culture medium was replaced with fresh, antibiotics-
free medium. Cells were co-transfected with plasmids coding reporter and target genes
pDsRed2-N1 (15 ng per well; Clontech Laboratories, Inc.) and pBACE-GFP 24 (70 ng per well,
provided by Dr. Weihong Song, The University of British Columbia, Vancouver, Canada)
and siRNAs (final concentration 0.1 and 1.0 nM) using LipofectamineTM 2000 (Invitrogen),
according to the manufacturer protocol. After 48 h of incubation, the cells were washed
with PBS and lysed in NP-40 buffer. The cell lysates were used for fluorescence measure-
ments. Fluorescence values of enhanced green fluorescent protein (EGFP) and red fluores-
cent protein (RFP) were measured using a Synergy HT reader (BIO-TEK); data quantification
was performed using KC4 software. Excitation and emission wavelengths were as follows:
GFP λEx = 485/20 nm and λEm = 528/20 nm; RFP λEx = 530/25 nm and λEm = 590/30 nm.
The siRNA activity was calculated as the ratio of GFP to RFP fluorescence values, averaged
over eight parallel repetitions. The relative level of fluorescence (GFP/RFP) in control cells
(transfected with pBACE-GFP, pDsRed2-N1 and control, non-silencing siRNA) was taken as
the reference (100%).
The work was supported by PBZ-MNiSW-07/I/2007 for years 2008–2010 to B. N. and by statutory
funds from Technical University of Lodz and Centre of Molecular and Macromolecular Studies of PAS.
The authors are grateful to Mr. M. Nowak for recording MALDI-TOF mass spectra, and Dr. P. Guga
and Prof. W. J. Stec for critical reading of the manuscript.
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