Activity of siRNAs with 2-thio-2'-O-methyluridine modification in mammalian cells

Article abstract of DOI:10.1080/15257770903316145

In a search to identify chemical modifications to improve the properties of siRNA, we have investigated the effect of the 2'-O-methyl-2-thiouridine modification on the biological activity of siRNA. Our results indicate that judicious placement of 2'-O-met

Full text of DOI:10.1080/15257770903316145

Nucleosides, Nucleotides and Nucleic Acids, 28:902–910, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903316145
ACTIVITY OF siRNAs WITH 2-THIO-2ꢀ-O-METHYLURIDINE
MODIFICATION IN MAMMALIAN CELLS
Thazha P. Prakash, Nishant Naik, Namir Sioufi, Balkrishen Bhat, and
Eric E. Swayze
Department of Medicinal Chemistry, Isis Pharmaceuticals Inc., Carlsbad, California, USA
In a search to identify chemical modifications to improve the properties of siRNA, we have
2
investigated the effect of the 2 ꢁ-O-methyl-2-thiouridine modification on the biological activity of
siRNA. Our results indicate that judicious placement of 2 ꢁ-O-methyl-2-thiouridine residues could
lead to modified siRNA with activity in mammalian cells.
Keywords siRNA; 2ꢁ-O-methyl-2-thiouridine; mRNA expression; oligonucleotides
Regulation of mRNA expression using small interfering RNAs (siRNAs)
has been recognized as a valuable research tool as well as a novel principle
for drug development.[1,2] The success of using short synthetic 19–21 RNA
duplexes to inhibit gene expression led to tremendous interest in develop-
ing this technology for the potential treatment of various diseases.[3,4]
For effective delivery to therapeutically relevant tissues after systemic
administration, chemical modifications are needed to improve the biodis-
tribution and pharmacokinetic properties of the siRNA. Several chemically
modified siRNAs have shown improved activity in cell culture as well as in
animal models.[3,5,6]
Oligonuleotides containing 2-thiouridine (s2U) residues are known
ꢁ
to favor C3 - endo sugar puckering for the nucleotides with an increase
in the rigidity of the backbone.[7] They also form thermodynamically
stable duplexes with complementary RNA.[8] Furthermore, the increased
size and weaker H-bonding ability of sulfur destabilizes a s2U -G wobble
and s2U-2-amino-A base pairs compared to uridine.[9] As a result, a s2U
containing oligonucleotide provides better selectivity toward binding to
complementary oligonucleotides. The RNA duplexes with 2ꢁ-O-methyl-2-
thiouridine (2ꢁ-O-Me-s2U) residues exhibited improved Tm relative to RNA
Received 4 April 2009; accepted 18 August 2009.
Address correspondence to Thazha P. Prakesh, Department of Medicinal Chemistry, Isis Pharma-
ceuticals Inc., Carlsbad, CA92008. E-mail: tprakashn@isisph.com
902

siRNAs with Thio-2ꢁ-O-methyluridine Modification
903
FIGURE 1 2ꢁ-O-Methyl and 2ꢁ-O-Me-2-thio modified RNA.
duplex.[10] Tm of RNA-RNA duplex containing 2ꢁ-O-Me- s2U was 5.5◦C per
modification higher than wild type duplex.[10] The oligonucleotides with
2ꢁ-O-MOE-2-thiothymidine residues exhibited enhanced binding affinity to
target RNA and stability to exonucleases compared to corresponding 2ꢁ-O-
MOE modified oligonucleotides.[11] Recently, it has been reported that s2U
residues suppress recognition of RNA by toll-like receptors.[12]
As a part of our ongoing effort in identifying chemical modifications[6]
to improve the properties of siRNA, we have evaluated the 2ꢁ-O-Me-s2U
modified (Figure 1) siRNA in cell culture. First, we have synthesized 2ꢁ-O-Me-
s2U-3ꢁ-[2-cyanoethyl-N ,N -diisopropyl]phosphoramidite 6 (Scheme 1) ac-
cording to the reported procedure.[10,11] 3ꢁ-O-Acetyl-5ꢁ-O-methanesulfonyl-
2ꢁ-O-methyluridine 2 was prepared from compound 1 according to stan-
dard procedures.[11] Compound 2 was dissolved in absolute ethanol and
refluxed in the presence of NaHCO3 to yield 2ꢁ-O-Me-2-O-ethyluridine 3
(80%). Compound 3 was stirred with 4,4ꢁ-dimethoxytrityl chloride (DMTCl)
in anhydrous pyridine to afford the 5ꢁ-O-DMT derivative 4. Treatment
of compound 4 with H2S in anhydrous pyridine in presence of 1,1,3,3-
tetramethylguanidine (TMG) yielded 5ꢁ-O-DMT-2ꢁ-O-Me-s2U 5 in 82% iso-
lated yield. Compound 5 was phosphitylated at the 3ꢁ-position to afford
phosphoramidite 6.
The guide strand (As, Table 1, 7) of the siRNAs used in this study were
designed to target the coding region of human PTEN mRNA.[13] Corre-
sponding guide strands containing 2ꢁ-O-Me and 2ꢁ-O-Me-s2U modifications
10–15 (Table 1) were designed and synthesized. The oligonucleotides 7–15
were synthesized using commercially available 2ꢁ-O-(tert-butyldimethylsilyl)
(2ꢁ-O-TBDMS) protected ribonucleoside phosphoramidites and 2ꢁ-O-Me
modified phosphoramidites on a solid phase oligonucleotide synthesizer
according to the reported procedure.[6] The phosphoramidite 6 was used
for the incorporation of 2ꢁ-O-Me-s2U residues in oligonucleotides 13–15
(Table 1). Oligonucleotides were characterized by mass spectroscopic (ES

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T. P. Prakesh et al.
SCHEME
1
a(i) (a) HOAc/H2O (4:1), room temerpature, (b). methanesulfonyl chloride,
pyridine:CH2Cl2 (1:1), −20◦C; (ii) NaHCO3 (2.5 mol eq.)/EtOH, reflux, 36 hours; (iii) DMT-Cl,
pyridine, room temerpature; (iv) H2S, TMG (10 mol eq.), pyridine, room temperature, 72 hours (v)
2-cyanoethyl N ,N ,N ꢁ,N ꢁ-tetraisopropylphosphorodiamidite, N ,N -diisopropyl ammonium tetrazolide,
CH3CN, room temperature.
MS) analysis (Table 1) and the purity was assessed by high performance
liquid chromatography (HPLC).
The siRNA duplexes were formed by combining equivalent molar
amount of As and passenger (S, Table 1, 8) strands mixed and heated at
TABLE 1 2ꢁ-O-Methyl and 2-thio-2ꢁ-O-methyl modified RNA used for this study
Calcd.
mass
No.
Sequence$
Found mass
7
8
9
10
11
12
13
14
15
5ꢁ r(UU UGU CUC UGG UCC UUA CUU) 3ꢁ
5ꢁ r(AA GUA AGG ACC AGA GAC AAA) 3ꢁ
6276.7
6507.2
6581.9
6544.2
6544.2
6544.2
6576.2
6560.1
6560.1
6275.8
6506.1
6580.3
6543.4
6543.7
6543.1
6575.2
6559.2
6559.2
5 ‘r(UU UGU CUC UGG UCC UUA CUU) 3ꢁ
5ꢁ r(UU UGU CUC UGG UCC UUA C∇ U∇ U∇ ) 3ꢁ
5ꢁ r(UU UGU CUC U∇ G∇ G∇ UCC UUA CUU) 3ꢁ
5ꢁ r(UU UGU C∇ U∇ C∇ UGG UCC UUA CUU) 3ꢁ
5ꢁ r(UU UGU CUC UGG UCC UUA C∇ U∗U∗) 3ꢁ
5ꢁ r(UU UGU CUC U∗G∇ G∇ UCC UUA CUU) 3ꢁ
5ꢁ r(UU UGU C∇ U∗C∇ UGG UCC UUA CUU) 3ꢁ
$Backbone chemistry: phosphorothioates, underline represents phosphodiester nucleotides, U∗
= 2ꢁ-O-methyl-2-thiouridine, U∇ = 2ꢁ-O-methyluridine, A∇ = 2ꢁ-O-methyl adenosine, C∇ = 2ꢁ-O-
methylcytidine, G∇ = 2ꢁ-O-mehtylguanosine.

siRNAs with Thio-2ꢁ-O-methyluridine Modification
905
TABLE 2 Activity of 2-thio-2ꢁ-O-methyl modified and 2ꢁ-O-methyl modified siRNA in inhibition of
endogenous PTEN mRNA in HeLa cells
siRNA
7:8
As 5ꢁ-3ꢁ/S 3ꢁ-5ꢁ
IC50 nM
0.2
5ꢁ r(UUUGUCUCUGGUCCUUACUU) 3ꢁ
3ꢁ r(AAACAGAGACCAGGAAUGAA) 5ꢁ
5ꢁ r(UUUGUCUCUGGUCCUUACUU) 3ꢁ
3ꢁ r(AAACAGAGACCAGGAAUGAA) 5ꢁ
5ꢁ r(UUUGUCUCUGGUCCUUAC∇ U∇ U∇ ) 3ꢁ
3ꢁ r(AAACAGAGACCAGGAAUGAA) 5ꢁ
5ꢁ r(UUUGUCUCU∇ G∇ G∇ UCCUUACUU) 3ꢁ
3ꢁ r(AAACAGAGACCAGGAAUGAA) 5ꢁ
5ꢁ r(UUUGUC∇ U∇ C∇ UGGUCCUUACUU) 3ꢁ
3ꢁ r(AAACAGAGACCAGGAAUGAA) 5ꢁ
5ꢁ r(UUUGUCUCUGGUCCUUAC∇ U∗U∗) 3ꢁ
3ꢁ r(AAACAGAGACCAGGAAUGAA) 5ꢁ
5ꢁ r(UUUGUCUCU∗G∇ G∇ UCCUUACUU) 3ꢁ
9:8
1.8
10:8
11:8
12:8
13:8
14:8
15:8
4.3
2.1
1.2
4.6
7.1
3ꢁ r(AAACAGAGACCAGGAAUGAA) 5ꢁ5ꢁ
r(UUUGUC∇ U∗C∇ UGGUCCUUACUU) 3ꢁ
3ꢁ r(AAACAGAGACCAGGAAUGAA) 5ꢁ
2.5
S = passenger strand; As = guide strand. Underline indicate phosphodiester linkages. U∗ = 2ꢁ-O-
methyl-2-thiouridine, U∇ = 2ꢁ-O-methyluridine A∇ = 2ꢁ-O-methyladenosine, C∇ = 2ꢁ-O-methylcytidine,
G∇ = 2ꢁ-O-mehtylguanosine.
90◦C for 1 minute. The solution was incubated at 37◦C for 6 hours. The
percentage of the duplex formed was determined using CGE analysis.[6]
Synthetic siRNA duplexes were transfected into cells using Lipofectin
reagent. Cells were harvested 18–24 hours posttransfection and total cellular
RNA was isolated on an RNeasy 3000 BioRobot (Qiagen, Valencia, CA,
USA). Reduction of target mRNA expression was determined by real
time quantitative RT-PCR. Total RNA for each well was measured using
RiboGreen[6] (Molecular Probes, Eugene, OR, USA), and these values were
used for sample-to-sample normalization.
The siRNA duplex 9:8 (Table 2) with phosphorothioate (PS) linkages
on the guide strand, exhibited dose dependent inhibition of PTEN mRNA
expression in cell culture (IC50 1.8 nM, Table 2). The siRNA duplex 7:8
(Table 2, IC50 0.2 nM) with the phosphodiester backbone chemistry (PO)
on the guide strand, showed 9 fold better activity.[6b] In this study, we have
used As strands with PS and S strand with PO linkages, respectively.
In our earlier report, we have evaluated the positional preference of
2ꢁ-O-Me modification on the activity of siRNA.[6b] The siRNA with three
2ꢁ-O-Me modified residues at the 3ꢁ-end of the As strand was more active than
siRNA with three 2ꢁ-O-Me modification at the 5ꢁ end of the As strand.[6b]
The activity of the siRNAs with and without 2ꢁ-O-Me modifications in the S
strands was similar.[6bb] Here we have evaluated the effect of replacing 2ꢁ-
O-Me U with 2ꢁ-O-Me-s2U on cellular activity of siRNA. In general, replacing

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T. P. Prakesh et al.
2ꢁ-O-Me-U with 2ꢁ-O-Me-s2U residue is tolerated in the As strand. siRNA
duplex 10:8 (IC50 4.3 nM, Table 2) containing 2ꢁ-O-Me U residues and 13:8
containing 2ꢁ-O-Me-s2U (IC50 4.6 nM, Table 2) residues at the 3ꢁ-end of
the As strand exhibited similar potency. However, when the 2ꢁ-O-Me-s2U
was placed in the middle of the As strand (Table 2, 14:8 IC50 7.1 nM,
15:8, IC50 2.5 nM), the activity was less than the corresponding 2ꢁ-O-Me U
siRNA (Table 2, 11:8 IC50 2.1 nM and 12:8 IC50 1.2 nM). These data suggest
that 2ꢁ-O-Me-s2U is well tolerated at the 3ꢁ-end of the As strand and 2-thio
base modification could maintain the activity of siRNA containing tandem
2ꢁ-O-Me modifications.
In conclusion, we have designed and synthesized siRNA duplexes with
2ꢁ-O-Me-s2U residues in the As strand. 2-Thio base modification in combina-
tion with 2ꢁ-O-Me sugar modification was well tolerated at the 3ꢁ-end of the
As strand of siRNA. The known nuclease resistant properties[11] of 2-thio
modification in conjunction with 2ꢁ-sugar modification, and the reported
results of 2-thiouridine suppress RNA recognition by toll-like recepotors[12]
suggests that this could be a useful modification for siRNA therapeutics
when used in combination with other modifications in the appropriate
substitution pattern.
EXPERIMENTAL
General
Reagents and phosphoramidites for oligonucleotide synthesis were pur-
chased from Glen Research, Inc. (Sterling, VA, USA). All other reagents
and anhydrous solvents were purchased from Sigma-Aldrich (St. Louis,
1
MO, USA) and used without further purification. H NMR spectra were
referenced using internal standard (CH3)4Si and 31P NMR spectra were
referenced using external standard 85% H3PO4. Mass spectra were recorded
by College of Chemistry, University of California (Berkeley, CA, USA).
5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ-O-methyl-2-thiouridine 5
Compound 4 (2.72 g, 4.38 mmol) was placed in a RB flask under an
argon atmosphere and cooled in a freezing bath. A solution of anhydrous
TMG (2.93 mL, 23.25 mmol) in pyridine (57 mL) was flushed with an
argon and cooled to 0◦C in a freezing bath and solution was saturated with
hydrogen sulfide for 45 minutes. The solution was then transferred into
the precooled flask containing compound 5 under an argon pressure. The
temperature of the flask was slowly brought up to room temperature and
stored for 72 hours. H2S was gently flushed out into a Clorox bath and
then pyridine was removed from the reaction mixture under vacuum. The
residue suspended in ethyl acetate was subjected to water wash followed

siRNAs with Thio-2ꢁ-O-methyluridine Modification
907
by standard workup. The desired product was purified by flash silica gel
column chromatography using ethyl acetate and hexane (1:1) as eluent
1
to yield compound 5 as a white foam (2.04 g, 82%): H NMR (200 MHz,
DMSO-d6) δ 3.33–3.49 (m, 2H), 3.51 (s, 3H) 3.73 (s, 6H), 3.80–3.85 (m,
1H), 4.01 (m, 1H), 4.30 (q, J = 7.6, 4.8, 7.4 Hz, 1H), 5.29 (d, J = 7.2 Hz,
1H), 5.39 (d, J = 8.2 Hz, 1H), 6.52 (d, J = 2.0 Hz, 1H), 6.91 (d, J = 8.8
Hz, 4H), 7.22–7.41 (m, 9H), 7.99 (d, J = 8.2 Hz, 1H), 12.71 (s, 1H); HRMS
(FAB) m/z calcd for C31H33N2O7S+ 577.2007, found 577.2008.
5ꢁ-O-(4,4ꢁ-Dimethoxytrityl)-2ꢁ-O-(methyl)-2-thiouridine-3ꢁ-[(2-
cyanoethyl)-N,N-diisopropyl]phosphoramidite 6
Compound 5 (1.14 g, 1.98 mmol) was mixed with tetrazole di-
isopropylammonium salt (0.17 g, 0.99 mmol) and dried over anhy-
drous P2O5 under reduced pressure overnight. The mixture was sus-
pended in anhydrous acetonitrile (7 mL) and 2-cyanoethyl-N ,N ,Nꢁ,Nꢁ-
tetraisopropylphosphordiamidite (1.26 mL, 3.98 mmol) was added. The
reaction mixture was stirred at the ambient temperature for 7 hours under
an argon atmosphere. The solvent was removed under reduced pressure
and the residue obtained was dissolved in ethyl acetate (20 mL), washed
with saturated aqueous sodium bicarbonate followed by standard workup.
The residue was purified by flash silica gel column chromatography (eluent:
ethyl acetate / hexane, 1:1) to yield compound 6 (1.11 g, 65%). 31P NMR
(80 MHz, CDCl3) δ 151.36; HRMS (FAB) m/z calcd for C40H50N4O8PS+
777.3087, found 777.3098.
Synthesis of Oligonucleotides 7–15
The standard phosphoramidites and solid supports were used for in-
corporation of A, U, G, and C residues. A 0.1 M solution of the phospho-
ramidites in anhydrous acetonitrile was used for the synthesis. The modified
oligonucleotides were synthesized on universal solid support.[14,15] A 0.1
M solution of phosphoramidite 6 was used for the incorporation of 2-
O-Me-s2U residues. Twelve equivalents of phosphoramidite solutions were
delivered in two portions, each followed by a 6 minutes coupling wait time.
All other steps in the protocol supplied by the manufacturer were used
without modification. 0.2 M PADS in 1:1 3-picoline/CH3CN were used as
a sulfurization reagent with 3 minutes contact time.[16] A solution of tert-
butyl hydroperoxide/acetonitrile/water (10:87:3) was used to oxidize inter
nucleosidic phosphite to phosphate. The step-wise coupling efficiencies
were more than 97%. After completion of the synthesis, the solid support
bearing oligonucleotide was suspended in aqueous ammonia (28–30 wt%):
ethanol (3: 1) and heated at 55◦C for 6 hours to complete the removal of all

908
T. P. Prakesh et al.
protecting groups except TBDMS group at 2ꢁ-position. The solid support
was filtered and the filtrate was concentrated to dryness. The residue
obtained was resuspended in triethylamine trihydrofluoride/triethylamine/
1-methyl-2-pyrrolidinone solution (0.75 mL of a solution of 1 ml of tri-
ethylamine trihydrofluoride, 750 µl triethylamine and 1.5 mL 1-methyl-2-
pyrrolidine, to provide a 1.4 M HF concentration) and heated at 65◦C for
1.5 hours to remove the TBDMS groups at the 2ꢁ-position. The reaction
was quenched with 1.5 M ammonium bicarbonate (0.75 mL) diluted with
DNase/RNase free water and purified by HPLC on a strong anion exchange
column (Mono Q, Pharmacia Biotech, 16/10, 20 mL, 10 µm, ionic capacity
0.27–0.37 mmol mL−1, A = 100 mM ammonium acetate, 30% aqueous
acetonitrile, B = 1.5 M NaBr in A, 0–60% B in 40 minutes, flow 1.5
mL min−1, λ = 260 nm). Fractions containing full-length oligonucleotides
were pooled together (assessed by LC MS analysis >90%) and evaporated.
The residue was desalted by HPLC on a reverse phase column (HPLC,
C-18 column, Waters, 7.8 × 300 mm, A = 100 mM ammonium acetate,
B = acetonitrile, C = water, 100% A two column volume, 100% C two
column volume, eluted oligonuclotide with 50% B in water, flow 2.5 mL
min−1, λ 260 nm). The oligonucleotides were characterized by ES MS and
purity was assessed by HPLC (Waters, C-18, 3.9 × 300 mm, A = 100 mM
triethylammonium acetate, pH = 7, B = acetonitrile, 5 to 60% B in 40 min,
flow 1.5 mL min−1, λ = 260 nm).
siRNA Preparation
The siRNA duplexes were prepared according to the reported
procedure.[6]
Cell Culture and Transfection of Cells
HeLa cells (American Type Tissue Culture Collection, Manassas, VA,
USA) were cultured in culture flask in Dulbecco’s Modified Eagle Medium
(DMEM, Invitrogen, CA, USA), liquid (high glucose) supplemented with
10% heat inactivated fetal bovine serum (FBS). The cells were not allowed to
exceed 75–80% confluency. Prior to treatment (24 h before treatment), the
cells were detached from the flask using Trypsin (Invitrogen) and plated in
96-well plates at a density of 5,000 cells/well. The cells were transfected with
siRNAs complexed with 6 µg mL−1 Lipofectin (Invitrogen) in serum-free
Opti-MEM I Reduced-Serum Medium. The cells were incubated in the
transfection medium for 4 hours, and transfection medium was removed
from the cells and replaced with fresh DMEM, 10% fetal calf serum and
incubated at 37◦C, 5% CO2 for 16 hours.

siRNAs with Thio-2ꢁ-O-methyluridine Modification
RNA Expression Analysis
909
Total RNA was harvested after 16 hours using the RNeasy pro-
cess from Qiagen according to the manufacturer’s protocol. Gene ex-
pression was determined via real time quantitative RT-PCR on the
ABI Prism 7900 system (Applied Biosystems, Foster City, CA, USA)
as described in the literature.[17–18] The following primer probe
set (Qiagen) was used: hu PTEN (Accession No. U92436.1), for-
ward primer 5ꢁ-AATGGCTAAGTGAAGATGACAATCAT, reverse primer
5ꢁ- TGCACATATCATTACACCAGTTCGT and FAM/TAMRA probe 5ꢁ-
TTGCAGCAATTCACTGTAAAGCTGGAAAGG. Total RNA for each well was
measured using RiboGreen (Molecular Probes), and these values were used
for sample-to-sample normalization.[19] IC50 values were calculated from the
linear regression analysis from the plot of the logarithmic value of siRNA
concentration versus percentage of untreated control.
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