Building oligonucleotide therapeutics using non-natural chemistries

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


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http://dx.doi.org/10.1016/j.cbpa.2006.10.001

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].


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-


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


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



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-


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


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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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.

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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


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.

39.�

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.

40.�

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.

41.�

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.

43.�

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.

45.�

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


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.


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.


Building oligonucleotide therapeutics using non-natural chemistries

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