Building oligonucleotide therapeutics using non-naturalchemistriesCharles Wilson and Anthony D Keefe
Modified nucleotides are increasingly being utilized in all
categories of therapeutic oligonucleotides to increase
nuclease-resistance, target affinity and specificity. The extent
to which these substitutions are tolerated varies with the
different modes of action exploited by various modalities, but
fully modified oligonucleotides have now been discovered for
most types of therapeutic oligonucleotide. Fully
phosphorothioate-substituted antisense oligonucleotides have
been used for several years. The first fully modified siRNA was
reported in 2006 with a 20-O-methyl sense strand and a
phosphorothioate antisense strand. The first fully modified
aptamer (20-O-methyl) was reported in 2005. It is expected that
future candidate therapeutic oligonucleotides will have even
more drug-like characteristics as a result of the inclusion of
modified nucleotides.
Addresses
Archemix Corp., 300 Third Street, Cambridge, MA 02142, USA
Corresponding author: Keefe, Anthony D ([email protected])
Current Opinion in Chemical Biology 2006, 10:607–614
This review comes from a themed issue on
Biopolymers
Edited by Hagan P Bayley and Floyd Romesberg
Available online 16th October 2006
1367-5931/$ – see front matter
# 2006 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.cbpa.2006.10.001
IntroductionOligonucleotides are becoming increasingly important as
candidate therapeutics as different modes of action are
discovered and developed. From the beginning of 2004
through mid-2006, several significant milestones were
achieved in this field. The first therapeutic aptamer
was approved by the FDA for age-related macular degen-
eration in December 2004, and marketed in 2005. The
first human clinical trial of an siRNA was initiated in 2004,
and antisense therapeutics have continued to progress
through all stages of clinical development. Virtually all
therapeutic oligonucleotides contain some fraction of
non-natural nucleotides. The driving factors for inclusion
of these modifications vary depending upon the mode of
action. A common factor is the desire to increase stability
by improving resistance to serum nucleases. Nuclease
degradation can be blocked by several means including
base, sugar and phosphate modifications (which prevent
endonuclease and exonuclease attack) as well as caps at
www.sciencedirect.com
the 30- and 50-termini (preventing attack by exonu-
cleases). Modifications may also be introduced into can-
didate therapeutic oligonucleotides to increase target
affinity and biological potency, to control biodistribution
(including intracellular uptake), and to facilitate synth-
esis. Appropriate choice of chemistries can simulta-
neously resolve a range of problems including affinity
for undesired targets, the propensity of certain oligonu-
cleotide motifs to self-aggregate, and potential toxicities.
In all instances, chemical modifications must be limited to
those that do not significantly inhibit activity — depend-
ing upon the mode of action of the oligonucleotide
therapeutic and the particular context of the modification,
different types of chemistries can be tolerated. In the
following sections, we review the impact of chemical
modifications on therapeutic oligonucleotides described
in the period from January 2004 until July 2006. Many of
the modified nucleotides discussed in this review are
shown in Figure 1.
AntisenseThe antisense therapeutic field is experiencing a renais-
sance with the introduction of successive generations of
nucleotide modifications that improve the drug-like char-
acteristics of these molecules. Over the past 15 years,
several base, sugar and phosphate changes have been
identified and shown to significantly reduce nuclease-
degradation rates while simultaneously increasing the
efficiency of target mRNA hybridization. Although most
antisense approaches have been designed to optimize
RNase H-mediated degradation of the targeted mRNA,
antisense oligonucleotides can also function via other
mechanisms. Simple hybridization to pre-mRNAs can
alter or prevent their splicing while hybridization to
processed mRNAs can block translation through changes
in ribosome loading and read-through.
Several recent papers describe clinical results with ‘first
generation’ antisense molecules — deoxyoligonucleo-
tides in which all phosphate linkages are replaced with
phosphorothioates. The antisense oligos G3139, also
known as Genasense or oblimersen, and ISIS 2302, also
known as Alicaforsen, have recently completed phase III
and phase II trials, respectively. G3139 is an 18-mer
oligodeoxynucleotide that targets the initiation codon
of the bcl-2 gene and has been studied as a co-treatment
for various cancers including leukemia, multiple mye-
loma, non-Hodgkin’s lymphoma, breast, prostate, and
small-cell lung cancer. Efficacy has been observed for
many of these indications although the clinical data to
date has failed to support regulatory approval. Debate
Current Opinion in Chemical Biology 2006, 10:607–614
608 Biopolymers
Figure 1
Modified nucleotides described in the text.
Current Opinion in Chemical Biology 2006, 10:607–614 www.sciencedirect.com
Building oligonucleotide therapeutics using non-natural chemistries Wilson and Keefe 609
continues over the precise mechanism of action of this
drug [1]. ISIS 2302, a 20-mer deoxyoligonucleotide tar-
geted to ICAM-1 mRNA, has recently completed a
clinical trial in ulcerative colitis [2]. Administered by
enema, this oligonucleotide showed quantitatively sig-
nificant and long-lasting improvement in patients includ-
ing mucosal healing and decreases in rectal bleeding.
Phase III trials are currently planned.
Since first-generation molecules were developed and
subsequently progressed into clinical development, sev-
eral additional chemistries have been evaluated for their
utility in an antisense setting. The current state-of-the-art
is represented by ‘gapmers’, oligonucleotides that typi-
cally contain phosphorothioate linkages throughout their
length and 20-modifications (e.g. 20-O-methyl, 20-O-meth-
oxyethyl) in both their 50-and 30-terminal portions. Both
types of chemical modifications increase nuclease-resis-
tance. 20-Modifications additionally facilitate stronger
base-pairing with target but are incompatible with RNase
H attack. Their omission from the central portion of the
gapmer allows the oligonucleotide to efficiently and
selectively pair with a target mRNA while preserving
its ability to induce target cleavage at the centre of the
duplex. ISIS 113715 is a representative gapmer that
targets PTP-1B, a key mediator of insulin resistance.
In a recently completed phase II trial with type II
diabetes patients, ISIS 113715 demonstrated statistically
significant improvement in multiple measures of glucose
control. Several other gapmers are currently in phase I and
phase II trials. A side benefit of the increased base-pair
strength obtained with the 20-MOE modifications is
increased binding specificity, thereby reducing toxicity
effects mediated by off-targets [3,4�,5].
Other sugar modifications include the bicyclic sugars
LNA (locked nucleic acid), ENA (ethylene-bridged
nucleic acid) and oxetane-modified ribose. Each of these
modifications has the effect of fixing the sugar conforma-
tion, thereby reducing the entropic cost of base-pairing
and increasing the thermodynamic stability of duplexes.
In a study of antisense activity against the oncogene H-
Ras, LNA-DNA-LNA gapmers showed the highest effi-
cacy among several different tested compositions in its
ability to inhibit tumour-growth in xenograft models.
Doses as low as 500 mg/kg/day demonstrated good effi-
cacy [6�]. In another study, ENA antisense oligonucleo-
tides were used to induce alternative splicing (specifically
exon-skipping) as a means for correcting expression of a
dystrophin mutation implicated in Duchenne muscular
dystrophy [7]. Oxetane-modified nucleotides confer
advantages when incorporated into antisense oligonu-
cleotides such as increased nuclease-resistance, target
affinity and specificity while supporting RNase H activity
and have been shown to be more potent than their
phosphorothioate equivalents when targeted to c-myb to
reduce gene expression [8].
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In another departure from natural sugars, substitution of
the ribose ring oxygen with a nitrogen and a carbon
(yielding a six-membered azasugar) has been shown to
greatly increase the potency of anti-HIV antisense oligo-
nucleotides in cell-based in vitro assays [9]. Several groups
have utilized ‘morpholino’ antisense oligonucleotides in
which a six-membered ring replaces the ribose. Nucleo-
tides are linked using an uncharged phosphordiamidite
linkage. These oligomers are highly nuclease-resistant
and show high specificity. Published work shows their
utility for transcript manipulation in vivo [10] as well as
the inhibition of gene expression [11].
A more extreme variation on the sugar theme is repre-
sented by peptide nucleic acids (PNAs). PNA is
uncharged and highly nuclease-resistant and has been
utilized in many antisense experiments to reduce gene-
expression [12]. It has been shown to be much more
potent than the corresponding phosphorothioate oligo-
deoxynucleotides in its ability to inhibit reverse-tran-
scription [13]. The structural similarity between ribose
and the amino acid proline is exploited by Wickstrom
et al. [14] who show that oligomers containing a trans-4-
hydroxy-L-proline backbone are highly efficacious in the
knockdown of specific protein targets in zebrafish embryo
assays.
Yet another backbone chemistry that has been tested in
an antisense context is the thiophosphoramidate modifi-
cation in which a non-bridging oxygen is substituted for a
sulfur and a bridging oxygen is substituted for a nitrogen.
The antisense construct GRN163L utilizes this backbone
and has been shown to inhibit the growth of various types
of human cancer cells both in vitro and in vivo when
targeted to the RNA template region of human telomer-
ase [15].
Boranophosphate-modified constructs, in which a non-
bridging oxygen is replaced by the isoelectronic borane
group (-BH3�) have been examined as antisense agents.
Hall and co-workers have shown that oligonucleotides
incorporating these modifications are highly potent at
suppressing GFP expression in HeLa cells [16]. A single
30-methylphosphonate-modified internucleotide linkage
at the 30-terminus has been shown to greatly reduce
oligonucleotide degradation in 10% fetal calf serum [17].
AptamersAptamers are short oligonucleotides which fold into well-
defined three-dimensional architectures, thereby
enabling specific binding to molecular targets such as
proteins. These molecules are typically obtained using
the SELEX process from combinatorial libraries of tran-
scripts in a manner analogous to phage display. Most
aptamers developed for therapeutic applications have
relied extensively upon nucleotide modifications to
improve their properties. Conceptually these modifica-
Current Opinion in Chemical Biology 2006, 10:607–614
610 Biopolymers
tions can be introduced ‘pre-SELEX’, by their incorpora-
tion into the initial transcript libraries, and ‘post-SELEX’,
through site-specific engineering.
The most significant recent event in the therapeutic
aptamer field was the FDA approval of MacugenTM
(pegaptanib sodium) in December 2004 for the treatment
of age-related macular degeneration (AMD) [18��,19�].Macugen binds to and inhibits VEGF with a KD of 49 pM
[20]. Of its 27 nucleotides, all but two bear either 20-fluoro
or 20-O-methyl modifications. Macugen was discovered
using the SELEX process starting with a 20-ribo purine,
20-fluoro pyrimidine transcript library. Following isolation
of a VEGF-binding sequence and minimization to
remove extraneous nucleotides not required for binding,
all but two of the 20-ribonucleotides were substituted for
20-O-methyl nucleotides to increase its stability to endo-
genous nucleases (substitution of the remaining two
nucleotides results in a significant loss of activity). The
optimized molecule exhibits a long intraocular half-life in
primate studies, and human clinical studies suggest a
terminal half-life of 10 days.
The mixed composition of Macugen (including ribo,
fluoro, and methoxy modifications) presents a variety of
challenges with respect to efficient, cost-effective synth-
esis. In considering the broader opportunities for aptamer
therapeutics which include chronic systemic administra-
tion, compositions with improved synthesis, reduced cost,
and better in vivo stability are expected to have a major
impact. In response, several groups have developed meth-
ods by which modified nucleotides may be introduced into
the initial SELEX libraries. A key constraint in exploring
alternative modifications is the substrate specificity of the
relevant polymerases required for synthesis of the initial
library and for each round of enzymatic amplification
during the SELEX process. Along these lines, Chelliser-
rykattil and Ellington [21] report the discovery of variant
T7 RNA polymerases that will accept 20-OMe A, C and U
(but not G). Burmeister et al. [22�] report conditions under
which the Y639F [23] and Y639F/H784A [24] polymerases
will accept all four 20-O-methyl nucleotides. Using these
methods, Burmeister et al. were able to discover a fully 20-O-methyl aptamer to VEGF whose serum stability com-
pares favourably with that of Macugen.
Focusing on a different set of chemistries, Kato et al. [25�]describe the synthesis of 40-thiopyrimidines (U and C) and
their incorporation into transcripts by T7 RNA polymer-
ase. The 40-thio modification increases the stability of
transcripts 50-fold relative to natural RNA transcripts.
Using these libraries, Kato et al. successfully performed
SELEX and were able to discover an aptamer to thrombin.
The feasibility of a variety of other modified libraries for
SELEX has been demonstrated through parallel efforts
by several groups. Vaught et al. [26�] show that several
Current Opinion in Chemical Biology 2006, 10:607–614
amino-acid-inspired side-chains substituted at the 5-posi-
tion of UTP can be synthesized and subsequently tran-
scribed using T7 RNA polymerase. Kuwahara et al. [27�]synthesized similar 5-modified dUTP compounds and
showed that KOD-DNA polymerase (i.e. the enzyme
from Thermococcus kodakaraensis) efficiently catalyzes their
template-dependent polymerization. Jager et al. [28] have
described a similar system for introducing a variety of
modified bases into oligonucleotides using other DNA
polymerases.
An alternative approach to the discovery of nuclease-
resistant aptamers relies upon the fact that nucleases
are highly enantioselective. Nuclease-resistant aptamers
can thus be discovered by initially performing SELEX
against an enantiomer of the biological target (synthe-
sized using D-amino acids) and then subsequently synthe-
sizing a mirror-image aptamer containing exclusively L-
ribose nucleotides. The resulting ‘spiegelmer’ specifically
recognizes the biological enantiomer of the target but
retains the high nuclease-resistance intrinsic to L-ribose
nucleotides. Several recent papers report further progress
with the pre-clinical development of spiegelmers includ-
ing demonstration that a ghrelin spiegelmer ameliorates
obesity in diet-induced obese mice [29]. A spiegelmer
specific for calcitonin gene-related peptide-binding
(CGRP) is effective in reducing electrically induced
cranial blood flow in response to the peptide [30].
Purschke et al. [31] show that a vasopressin-binding
spiegelmer functions as a diuretic (aquaretic) in rats.
Paralleling the ‘pre-SELEX’ methods for aptamer stabi-
lization, new strategies for post-SELEX optimization
continue to be developed. Schmidt et al. [32] demon-
strated that LNA residues may be substituted into a
tenascin-C aptamer to increase the thermal stability
and nuclease-resistance of the aptamer. In common with
other aptamer substitution strategies, some substitutions
lead to a loss of target binding.
Decoy oligonucleotidesDecoy oligonucleotides are conceptually related to apta-
mers to the extent that they bind proteins directly,
competing with promoter elements for binding by tran-
scription factors, and thus altering the transcription of
targeted genes. As with other modalities, nucleotide
modifications may be introduced to modify the properties
of decoy oligos (e.g. cellular uptake, nuclease resistance)
but the benefits must be balanced against potential
changes in binding to the targeted transcription factor(s).
Crinelli et al. [33] introduced both D- and L-LNA mod-
ifications into a double-stranded decoy oligonucleotide
that binds to NF-kB and found positions where several
such modifications are tolerated. Timchenko et al. [34]
have generated a double-stranded decoy oligonucleotide
that irreversibly binds to the same target using a
reactive modified internucleotide linkage comprising a
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Building oligonucleotide therapeutics using non-natural chemistries Wilson and Keefe 611
trisubstituted pyrophosphate moiety. Phosphorothioates
have been successfully introduced into DNA duplexes
targeting the RNase H domain of HIV-1 [35] and NF-kB
dimers [36].
Immunostimulatory oligonucleotidesSome oligonucleotide sequences are specifically recog-
nized by pattern recognition receptors of the innate
immune system and thereby elicit responses such as
up-regulation of IL-6, interferon-a, and, interferon-g.
The archetypal motif in this class is the CpG dinucleo-
tide, which has been shown to activate Toll-like teceptor
9 (TLR9) as part of an ancient eukaryotic immune
response mechanism to bacterial DNA. Phosphorothio-
ate-modified oligonucleotides containing CpG motifs are
currently being evaluated in clinical trials for a range of
indications including cancer, infectious diseases, allergies
and as vaccine adjuvants where the immunostimulatory
response may have a therapeutic benefit. The phosphor-
othioate modification increases nuclease resistance with-
out significantly reducing the ability of the CpG-
containing oligonucleotide to elicit an immune response.
The most clinically advanced immunostimulatory oligo-
nucleotide is CPG7909 (also known as PF-3512676),
currently being evaluated as a combination therapy for
the treatment of multiple cancer types. CPG7909 has also
been tested as an adjuvant for several different vaccines
(see for example [37,38]). The effect of various base
modifications to the CpG motif on the immune response
has been investigated [39�]. Although most modifications
result in a loss of TLR9 stimulation, a handful including,
for example, the CpI (replacing guanosine with inosine)
support significant stimulation.
Small interfering RNASiRNAs are short duplexes designed to specifically trigger
the enzymatic destruction of specific transcripts via the
RNA interference pathway. A typical siRNA construct
contains a 21-nucleotide long RNA duplex with two base-
pair overhangs at each terminus. Unmodified siRNA
constructs are susceptible to degradation by RNases
and this is a major limitation to their efficacy. Work with
a variety of different modified oligonucleotides shows
that not all nuclease-stabilizing modifications are compa-
tible with loading the siRNA into the RNA-induced
silencing complex (RISC), a requirement for gene silen-
cing. The last few years have seen considerable efforts
expended to determine which nuclease-resistant modifi-
cations can be introduced into siRNA constructs without
reducing their potency. This progression is reflected in
the evolution of the siRNA constructs that are entering
clinical trials with early trials including entirely unmodi-
fied siRNA and more recent ones including modified
nucleotides.
Work from several laboratories has explored general and
context-specific effects of many of the standard stabiliz-
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ing oligonucleotide modifications (e.g. 20-O-methyl, 20-fluoro, 20-O-methoxyethyl and phosphorothioate). Kray-
nack and Baker [40�] reported functional constructs with
IC50 values in the low nanomolar range with a fully 20-O-
methyl sense strand and a fully phosphorothioate-mod-
ified antisense strand. Jackson et al. [41�] reported that
many individual nucleotides in the antisense strand may
be modified with 20-O-methyl groups while preserving
RNA interference effects upon the target, a similar study
has been performed with 20-fluoro, 20-O-methyl and 20-O-
MOE [42�]. An additional benefit of this modification is a
reduction in off-target effects as assessed by transcription
profiling. Similarly, Fedorov et al. [43�] showed that
siRNA constructs minimally modified with 20-O-methyl
groups had reduced off-target effects. Allerson et al. [44]
reported that siRNA constructs with alternating 20-O-
methyl and 20-fluoro nucleotides are very potent and
extremely stable. The first demonstration of the use of
an siRNA construct to silence a therapeutically relevant
gene in vivo was with a construct that contained a limited
number of both 20-O-methyl and phosphorothioate mod-
ifications clustered close to its 30-termini [45�]. Other
nucleotide modifications that have shown activity as
siRNAs include boranophosphates [46] and 40-thioriboses
[47].
DeliveryWith the exception of aptamers and immunostimulatory
molecules that interact directly with extracellular protein
targets, therapeutic oligonucleotides must enter cells to
be active. Their polyanionic nature intrinsically limits
permeation across the cell membrane. Strategies such as
reducing or eliminating charge by replacing the phospho-
diester linkage between nucleotides with uncharged mor-
pholino [48] or phosphono [49] linkers have been tested.
Alternatively, receptor-mediated routes into the cell can
be utilized as means for both facilitating cellular uptake
and limiting the subset of cells targeted with the oligo-
nucleotide. Along these lines, McNamara et al. [50�]describe recent results with a 20-ribopurine, 20-fluoropyr-
imidine oligonucleotide containing both a PSMA (pros-
tate-specific membrane antigen) aptamer and an siRNA
sense strand. After annealing with a synthetic siRNA
antisense strand, the construct was shown to induce
RNA interference only in cells expressing PSMA. This
elegant work demonstrates the ways in which different
types of modalities benefiting from a variety of modifica-
tion chemistries can be combined to yield molecules with
unique functional properties.
ConclusionConsiderable progress has been recently made in increas-
ing the drug-like characteristics of various oligonucleotide
therapeutic modalities by the introduction of modified
nucleotides. These characteristics include target affinity,
potency, stability, safety and ease of synthesis. Although
fully modified antisense (phosphorothioate) molecules
Current Opinion in Chemical Biology 2006, 10:607–614
612 Biopolymers
have been widely utilized for some time, recent efforts
have yielded the first demonstrations of highly activity,
fully modified aptamers (20-O-methyl) and siRNAs (20-O-
methyl and phosphorothioate) [22�,40�]. Continued
efforts to enable new chemistries and to test those che-
mistries in the variety of different contexts will continue
to improve the prospects for oligonucleotides as thera-
peutics.
AcknowledgementsSupport from Archemix Corp. is gratefully acknowledged by the authors.
References and recommended readingPapers of particular interest, published within the annual period ofreview, have been highlighted as:
� of special interest�� of outstanding interest
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19.�
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25.�
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26.�
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Amino acid-derived side chains are linked to the 5-position of dUTP andshown to support DNA synthesis catalyzed by the KOD DNA polymerase.These results set the stage for future SELEX experiments.
28. Jager S, Rasched G, Kornreich-Leshem H, Engeser M, Thum O,Famulok M: A versatile toolbox for variable DNAfunctionalization at high density. J Am Chem Soc 2005,127:15071-15082.
29. Shearman LP, Wang SP, Helmling S, Stribling DS, Mazur P, Ge L,Wang L, Klussmann S, Macintyre DE, Howard AD, Strack AM:Ghrelin neutralization by a ribonucleic acid-SPM amelioratesobesity in diet-induced obese mice. Endocrinology 2006,147:1517-1526.
30. Denekas T, Troltzsch M, Vater A, Klussmann S, Messlinger K:Inhibition of stimulated meningeal blood flow by a calcitoningene-related peptide binding mirror-image RNAoligonucleotide. Br J Pharmacol 2006, 148:536-543.
31. Purschke WG, Eulberg D, Buchner K, Vonhoff S, Klussmann S:An L-RNA-based aquaretic agent that inhibits vasopressinin vivo. Proc Natl Acad Sci USA 2006, 103:5173-5178.
32. Schmidt KS, Borkowski S, Kurreck J, Stephens AW, Bald R,Hecht M, Friebe M, Dinkelborg L, Erdmann VA: Applicationof locked nucleic acids to improve aptamer in vivo stability andtargeting function. Nucleic Acids Res 2004, 32:5757-5765.
33. Crinelli R, Bianchi M, Gentilini L, Palma L, Sorensen MD,Bryld T, Babu RB, Arar K, Wengel J, Magnani M:Transcription factor decoy oligonucleotides modifiedwith locked nucleic acids: an in vitro study to reconcilebiostability with binding affinity. Nucleic Acids Res 2004,32:1874-1885.
34. Timchenko MA, Rybalkina EY, Lomakin AY, Evlakov KI,Serdyuk IN, Ivanovskaya MG: Modified DNA fragmentsspecifically and irreversibly bind transcription factorNF-kappaB in lysates of human tumor cells. Biochemistry(Mosc) 2006, 71:454-460.
35. Somasunderam A, Ferguson MR, Rojo DR, Thiviyanathan V, Li X,O’Brien WA, Gorenstein DG: Combinatorial selection, inhibition,and antiviral activity of DNA thioaptamers targeting the RNaseH domain of HIV-1 reverse transcriptase. Biochemistry 2005,44:10388-10395.
36. Bassett SE, Fennewald SM, King DJ, Li X, Herzog NK,Shope R, Aronson JF, Luxon BA, Gorenstein DG: Combinatorialselection and edited combinatorial selection ofphosphorothioate aptamers targeting human nuclearfactor-kappaB RelA/p50 and RelA/RelA. Biochemistry 2004,43:9105-9115.
37. Cooper CL, Davis HL, Morris ML, Efler SM, Adhami MA, Krieg AM,Cameron DW, Heathcote J: CPG 7909, an immunostimulatory
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TLR9 agonist oligodeoxynucleotide, as adjuvant to Engerix-BHBV vaccine in healthy adults: a double-blind phase I/II study.J Clin Immunol 2004, 24:693-701.
38. Meng Y, Carpentier AF, Chen L, Boisserie G, Simon JM,Mazeron JJ, Delattre JY: Successful combination of localCpG-ODN and radiotherapy in malignant glioma. Int J Cancer2005, 116:992-997.
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Vollmer J, Weeratna RD, Jurk M, Davis HL, Schetter C,Wullner M, Wader T, Liu M, Kritzler A, Krieg AM: Impact ofmodifications of heterocyclic bases in CpG dinucleotideson their immune-modulatory activity. J Leukoc Biol 2004,76:585-593.
Immunostimulation via TLR9 is explored in different transfected celltypes. Of particular note, CpI motifs (in which inosine substitutes forguanosine) shows significant activity.
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Kraynack BA, Baker BF: Small interfering RNAs containingfull 20-O-methylribonucleotide-modified sense strandsdisplay Argonaute2/eIF2C2-dependent activity. RNA 2006,12:163-176.
Contradicting previous reports, the authors show that it is possible togenerate fully 20-O-methyl substituted siRNA sense constructs that retainfunctional activity.
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Jackson AL, Burchard J, Leake D, Reynolds A, Schelter J,Guo J, Johnson JM, Lim L, Karpilow J, Nichols K, Marshall W,Khvorova A, Linsley PS: Position-specific chemicalmodification of siRNAs reduces ‘‘off-target’’ transcriptsilencing. RNA 2006, 12:1197-1205.
20-O-methyl modifications at specific positions within an siRNA guidestrand are shown to reduce silencing of partially complementary tran-scripts while not affecting perfectly matched targets. The results suggestways in which chemical modifications can be used to limit the extent ofoff-target effects with siRNA.
42.�
Prakash TP, Allerson CR, Dande P, Vickers TA, Sioufi N, Jarres R,Baker BF, Swayze EE, Griffey RH, Bhat B: Positional effect ofchemical modifications on short interference RNA activity inmammalian cells. J Med Chem 2005, 48:4247-4253.
The sensitivity of siRNA modifications at the 20-position are exploredthrough a systematic study. By introducing 20-fluoro, 20-O-methyl, and 20-MOE modifications at discrete positions within sense and antisensestrands of an siRNA construct and then assessing their functional activityin Hela cells, structure–activity relationships to guide the design ofstabilized siRNAs can be inferred.
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Fedorov Y, Anderson EM, Birmingham A, Reynolds A, Karpilow J,Robinson K, Leake D, Marshall WS, Khvorova A: Off-targeteffects by siRNA can induce toxic phenotype RNA. 2006,12:1188-1196.
Appropriate chemical modifications are shown to prevent off-targeteffects on gene expression with siRNA constructs.
44. Allerson CR, Sioufi N, Jarres R, Prakash TP, Naik N, Berdeja A,Wanders L, Griffey RH, Swayze EE, Bhat BJ: Fully 20-modifiedoligonucleotide duplexes with improved in vitro potency andstability compared to unmodified small interfering RNA.Med Chem 2005, 48:901-904.
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Soutschek J, Akinc A, Bramlage B, Charisse K, Constien R,Donoghue M, Elbashir S, Geick A, Hadwiger P, Harborth Jet al.: Therapeutic silencing of an endogenous gene bysystemic administration of modified siRNAs. Nature 2004,432:173-178.
Chemically modified siRNAs targeting apo-B are shown to block targetexpression following systemic administration.
46. Hall AH, Wan J, Shaughnessy EE, Ramsay Shaw B, Alexander KA:RNA interference using boranophosphate siRNAs: structure-activity relationships. Nucleic Acids Res 2004, 32:5991-6000.
47. Hoshika S, Minakawa N, Kamiya H, Harashima H, Matsuda A:RNA interference induced by siRNAs modified with40-thioribonucleosides in cultured mammalian cells.FEBS Lett 2005, 579:3115-3118.
48. Takei Y, Kadomatsu K, Yuasa K, Sato W, Muramatsu T:Morpholino antisense oligomer targeting human midkine:its application for cancer therapy. Int J Cancer 2005,114:490-497.
49. Kocisova E, Praus P, Rosenberg I, Seksek O, Sureau F,Stepanek J, Turpin PY: Intracellular uptake of modified
Current Opinion in Chemical Biology 2006, 10:607–614
614 Biopolymers
oligonucleotide studied by two fluorescence techniques.Biopolymers 2004, 74:110-114.
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McNamara JO II, Andrechek ER, Wang Y, Viles KD, Rempel RE,Gilboa E, Sullenger BA, Giangrande PH: Cell type-specificdelivery of siRNAs with aptamer-siRNA chimeras.Nat Biotechnol 2006, 24:1005-1015.
Current Opinion in Chemical Biology 2006, 10:607–614
Chimeric molecules containing an aptamer specific for PSMA fused to ansiRNA construct targeting cell survival genes are prepared and tested.The presence of the PSMA aptamer specifically targets the siRNA fordelivery and uptake by prostate tumour cells, leading to improvedinhibition of tumour growth in xenograft models.
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