578
`
`Mini-Reviews in Medicinal Chemistry, 2010, 10, 578-595
`
`Recent Progress in Chemically Modified siRNAs
`
`M. Gaglione and A. Messere*
`
`Department of Environmental Sciences, Second University of Naples, via Vivaldi 43, 81100, Caserta, Italy
`
`Abstract: RNA interference technology has become a powerful laboratory tool to study gene function. Small interfering
`RNAs (siRNAs) have provided unprecedented opportunities for the development of new therapeutics in human diseases.
`Unfortunately, siRNA duplexes are not optimal drug-like molecules. The problems for their effective application are fun-
`damentally delivery, stability and off-target effects. Chemical modification provides solutions to many of the challenges
`facing siRNA therapeutics. In this review, we recapitulate and discuss the development of the latest described chemical
`modifications of siRNAs, with a special focus on novel chemical modifications of siRNA structure, architecture and
`siRNA conjugates.
`
`Keywords: RNAi, siRNAs, chemical modification, siRNA conjugates.
`
`INTRODUCTION
`
` The completion of human genome sequencing project
`and the elucidation of many molecular pathways that are
`important in diseases have made the use of nucleic acid-
`based inhibitors of gene expression an immense attractive-
`ness in functional genomics and therapeutic strategies. The
`increase in Oligonucleotide-Mediated Gene Silencing [1]
`(OMGS) is largely a result of improvements in the rational
`design of ONs and in the methods used for synthesizing oli-
`gonucleotides with relatively inexpensive cost. The concept
`underlying nucleic acids based technology is relatively
`straightforward: the use of a sequence that recognizes a spe-
`cific mRNA through Watson-Crick base pair hybridization
`inhibits gene expression at either the transcriptional or post-
`transcriptional level [2]. The major classes of inhibitory
`agents are Antisense Oligonucleotides (AS-ONs or ASOs)
`and more recently Small Interfering RNA (siRNAs).
`
`From Antisense to siRNA
`
` The notion that gene expression could be modified
`through the use of small ODNs derives from studies by
`Paterson et al. [3], who first used singlestranded DNA to
`inhibit translation of a complementary RNA in a cell-free
`system in 1977. The following year, Zamecnik and Stephen-
`son [1, 4] showed that a short (13-mer) DNA that was AN-
`TISENSE to the Rous sarcoma virus could inhibit viral rep-
`lication in culture. In the mid 1980s, the existence of natu-
`rally occurring antisense RNAs and their role in regulating
`gene expression was shown [5, 6]. Since then, the antisense
`strategy has enjoyed exponential gains in interest and has
`been the subject of thousands of published reports. The basic
`concept and the mechanisms of inhibition of AS-ONs have
`been characterized and discerned in two main mechanisms:
`steric-blockage and RNase-H activation
`[7, 8]. The
`
`*Address correspondence to this author at the Department of Environmental
`Sciences, Second University of Naples, via Vivaldi 43, 81100, Caserta,
`Italy; Tel: +39-(0)823-274602; Fax: +39-(0)823-274605;
`E-mail: anna.messere@unina2.it
`
`steric-blocker AS-ONs physically prevent or inhibit the pro-
`gression of splicing or the translation machinery, while the
`RNase H dependent oligonucleotides induce the degradation
`of mRNA. Most of the oligonucleotides capable of inhibiting
`splicing are not RNase-H dependent [9-11]. In spite of the
`theoretical simplicity, the molecular mechanism of action of
`AS-ONs has critical drawbacks such as poor stability versus
`nuclease activity in vitro and in vivo, low intracellular pene-
`tration and low bioavailability [12, 13]. Synthetic nucleic
`acids can be chemically modified to improve their general
`pharmacodynamic properties for their therapeutic use. Hun-
`dreds of different modifications have been studied for utility
`in antisense applications [14-16]. Three generations of
`chemically modified ASOs have been developed to enhance
`nuclease resistance, prolong tissue half-life, reduce off-target
`effects and increase affinity and potency [7, 17-24]. The
`most notable discovery was the introduction of a phos-
`phorothioate backbone in oligonucleotides, leading to a sig-
`nificant increase in their stability without major changes in
`ability to hybridize their target mRNA [7]. Other chemical
`modifications, including the development of DNA/RNA
`mixed-backbone oligonucleotides [25] and other oligonu-
`cleotides barely resembling DNA (such as peptide nucleic
`acid (PNA) and locked nucleic acid (LNA) structures) [7],
`have been made to increase the efficacy and stability of the
`antisense molecules. Although a few first generation ASOs
`have shown promising therapeutic performances, newer gen-
`erations have entered into clinical trials.
`
`RNA interference (RNAi) is the most recent explotion of
`interest in antisense world following the discoveries of Mello
`and colleagues [26] that double-stranded RNAs (dsRNAs)
`elicit potent degradation of targeted mRNA sequences in C.
`elegans and in mammalian cells [27, 28]. The active compo-
`nents of the RNAi are dsRNAs, small interfering RNA (siR-
`NAs), that tipically contain 19-21 bp and 2-nt 3’-overhangs.
`These short species are naturally produced by Dicer- medi-
`ated cleavage of larger dsRNAs and they are functional in
`mammalian cells. Synthetic siRNAs can also be introduced
`into cells exogenously in order to experimentally activate
`RNAi [27]. When an exogenous 19-21 bp siRNA is intro-
`
`1389-5575/10 $55.00+.00
`
`© 2010 Bentham Science Publishers Ltd.
`
`Alnylam Exh. 1032
`
`

`

`Recent Progress in Chemically Modified siRNAs
`
`Mini-Reviews in Medicinal Chemistry, 2010, Vol. 10, No. 7 579
`
`duced into a mammalian cell the 5’-end is phosphorylated.
`The duplex is assembled to form the RNA-Induced Silencing
`Complex (RISC), a multiprotein complex including Argo-
`naute 2 (AGO2), Dicer, TRBP (HIV-1 TAR RNA binding
`protein) and PACT (a dsRNA-binding protein), as well as
`other proteins, some of which are yet unknown [29]. The
`RISC proteins facilitate searching through the genome for
`RNA sequences that are complementary to one of the two
`strands of the siRNA duplex. One strand of the siRNA (the
`sense or passenger strand) is lost from the complex, while
`the other strand (the antisense or guide strand) is matched
`with its complementary RNA. In particular, the targets of
`siRNA-loaded RISC are mRNAs presenting a perfect se-
`quence complementarity. When siRNA-mediated silencing
`occurs, after the cleavage, release and degradation of the
`products, RISC-complex can interact with other molecules
`from the mRNA pool [30]. It was shown that endogenously
`expressed small RNA molecules, named microRNAs (miR-
`NAs), elicit gene-silencing in higher organisms [31, 32].
`MiRNAs are not involved in the pathway that leads to the
`production of a protein, instead they regulate the expression
`of mRNAs. The targets of miRNA-loaded RISC are mRNAs
`presenting a perfect sequence complementarity with nucleo-
`tides 2-7 in the 5’ region of the miRNA (the so-called seed
`region), and additional base pairings with its 3’-region. RISC
`mediates downregulation of gene expression by cleavage or
`translational inhibition of the target mRNA. The choice be-
`tween these two modes of action is dictated by the degree of
`complementarity between miRNA and its target. Near-
`perfect complementarity produces cleavage of the mRNA, as
`well as siRNAs, followed by its complete degradation,
`whereas partial complementarity causes translational inhibi-
`tion [33]. In vertebrates, most of the time, the consequence is
`an inhibition of translation. In addition to repressing transla-
`tion, miRNA interactions can promote the deadenylation or
`decapping, leading to rapid mRNA decay [34]. MicroRNA
`target sequences are usually located in the 3’-UTR of
`mRNAs. A single miRNA is expected to target at least 100
`transcripts from various genes, and one mRNA may be tar-
`geted, at its 3’-end, by different miRNAs [35]. In mammal-
`ian cells, long double strand RNAs (> 30 nt) induce immune
`responses [36] resulting in global gene silencing and in cer-
`tain cases cell death [37, 38]. This fact prevented the applica-
`tion of RNAi to mammalian cells until it was discovered that
`21-mer siRNA duplexes were capable of effecting potent and
`specific gene knockdown without inducing of the interferon
`response [27]. However, the immunogenic activity of short
`RNA duplexes is more significant than it was originally
`thought. Recent findings report off-target effects resulting
`from immunostimulation caused by dsRNAs that are shorter
`than 30 bp [39-46]. Immune response activation by short
`RNAs is a complex process. Briefly, RNAs are recognized
`by three main types of immunoreceptors: Toll-like receptors
`(specifically TLR3, TLR7 and TLR8), protein kinase R
`(PKR) and helicases such as RIG-I and MDA5. Recognition
`by these receptors can lead to a number of cellular responses
`including release of cytokines and changes in gene expres-
`sion [42]. The receptors involved are expressed on cell sur-
`faces (TLR3) [47], in endosomes (TLR3/7/8) [48-50] and in
`the cytoplasm (RIG-I, MDA5, PKR) [49]. Given its reliabil-
`ity and ease of use, RNAi has become the most widely used
`
`technology in functional genomic studies in vitro and in sev-
`eral model organisms. To translate this potential into a broad
`new family of therapeutics, it is necessary to optimize the
`efficacy of the RNA-based drugs. As discussed in this re-
`view, it might be possible to achieve this optimization using
`chemical modifications that improve, just like for ASOs,
`their in vivo stability, cellular delivery, biodistribution,
`pharmacokinetics, potency, and specificity. Previous experi-
`ence with antisense oligonucleotides is directly relevant to
`the clinical progress of siRNAs. Indeed, siRNAs and an-
`tisense oligonucleotides have more similarities than differ-
`ences. ASOs and siRNAs are natural phosphodiester com-
`pounds, they are short (~20 base) nucleic acids and are large,
`negatively charged molecules [51, 52]. Similar protocols
`exist for the large-scale synthesis of nucleic acid drugs in
`amounts needed for clinical trials. Such as ASOs, siRNAs do
`not readly cross the cellular membrane because of their nega-
`tive charge and size [53]. There are biological barriers that
`stand between initial administration of oligonucleotides and
`their final actions within cells. One the first biological barri-
`ers encountered by administered naked ASOs and siRNAs is
`represented by the nuclease activity in plasma and tissues.
`Single-stranded nucleic acids are rapidly degraded in serum
`or inside cells. Double-stranded nucleic acids, including
`siRNAs, are more stable than their single stranded counter-
`parts, but are still degraded and must be protected from nu-
`clease attack [54]. The systemic administration of ASOs and
`siRNAs results in primary localization to liver while their
`local administration is an option for many diseases. They
`have a common target: siRNAs are also intended to bind by
`Watson-Crick base-pairing complementary mRNA, and they
`can induce target RNA destruction. Both ASOs and siRNAs
`can incorporate chemical modifications in order to improve
`their in vivo properties. SiRNAs and ASOs, because of syn-
`thetic nature can be recognized as “foreign” by the innate
`immune system. In addition, for siRNAs both strands have
`the potential to trigger unwanted side effects [55 and cited
`references]. Finally, antisense and siRNA molecules are in-
`volved in ongoing testing in multiple clinical trials. There are
`also significant differences between antisense oligonucleo-
`tides and siRNAs. siRNAs contain two strands, which must
`be synthesized separately and then hybridized. Each strand
`has a molecular weight of approximately 7000 Da and the
`large size, as well as the charged backbone, discourage easy
`passage of RNA duplex through cell membranes. The target
`recognition of siRNA is mediated through the RISC com-
`plex. Moreover, very few molecules of siRNA are needed to
`inhibit gene expression [56]: siRNAs co-opt a natural silenc-
`ing pathway and the RISC proteins facilitate efficient recog-
`nition of target sequences by siRNAs, whereas ASOs must
`find their targets unassisted. Because endogenous duplex
`RNAs control important physiologic processes, introducing
`synthetic RNA may pertub this native machinery. ASOs
`have one strand that can block RNA by a steric mechanism
`or form a DNA-mRNA hybrid that recruits RNase H. Other
`mechanisms described for the ASOs include interference
`with mRNA processing and transport, and formation of a
`triplex directly with DNA into nucleus (Triplex Forming
`Oligonucleotides, TFO) [57, 58]. The mechanism by which
`antisenses exert their effects, largely depends on their struc-
`ture and chemistry: morpholino, PNA and 2’-O-alkyl modi-
`
`

`

`580 Mini-Reviews in Medicinal Chemistry, 2010, Vol. 10, No. 7
`
`Gaglione and Messere
`
`fied ASOs are capable of acting by other mechanisms e.g.
`they can inhibit intron excision, a key step in the processing
`of mRNA. Splicing occurs into nucleus during the matura-
`tion step of pre-mRNA and can be inhibited by hybridization
`of an oligonucleotide to the 5’ and 3’-regions involved in
`this process. ASOs can act like splicing regulator while siR-
`NAs cannot target nuclear RNAs or introns [59]. When
`chemical modifications are introduced into siRNAs it is im-
`portant to underline that modified siRNAs work through an
`only pathway and must interact with a number of different
`cellular proteins. Many or all of these proteins may be sensi-
`tive to changes in siRNA structure caused by the modifying
`group. SiRNA duplexes have been modified in a wide vari-
`ety of ways and the reported results seem to contradict one
`another.
`
` The better and recent understanding of both ASOs and
`siRNA properties have raised an important discussion about
`the future of antisense and siRNA in clinic applications. But
`the theoretical discussion of the relative value of siRNA and
`antisense oligonucleotides can only be resolved by clinical
`development. In fact, the widespread and rapid adoption of
`siRNA in cell culture, due to the potency of the natural
`mechanism of RNAi, has supported the conclusion that
`siRNA strategy has advantages for gene silencing respect to
`ASOs approach. However, further work will be needed to
`improve in vivo potency, biodistribution and toxicity of siR-
`NAs. Certainly the future of siRNA as therapy will involve
`advanced medicinal chemistry to improve both biodistribu-
`tion to tissue and uptake by specific cell types to reduce off-
`target effects and enhance the efficiency of silencing by
`those duplexes that ultimately reach target cells.
`
` A logical starting point for the reaching of this goal has
`lied with the previous generation of research on the proper-
`ties of ASOs. Because of this head start, progress that re-
`quired 10-15 years for antisense oligonucleotides has re-
`quired just 2-4 years for siRNA. The lesson learned from
`antisense drug clinical development has provided insights
`into how to choose target genes, into experimental pitfalls
`and the criteria for designing well-controlled experiments in
`drug based RNAi. In conclusion, the chemical modifications
`that have been developed to optimize the properties of
`ASOs, have had a major impact on the properties and effi-
`cacy of siRNAs.
`
`Functional Anatomy of siRNA: The Rationale for Chemi-
`cal Modification of siRNAs
`
` On the basis of analyses of a small number of silenced
`genes in mammalian cells, a set of empirical guidelines have
`already been proposed for siRNA design. These rules require
`the generation of RNA duplexes containing a 19-nt duplexed
`region, symmetric 2-3-nt 3’ overhangs, 5’-phosphate and 3’-
`hydroxyl groups targeting a region in the gene to be silenced.
`In most cases, only one of the two strands of a siRNA enters
`the RISC with high efficiency. This so-called guide strand is
`selected on the basis of thermodynamic bias and is responsi-
`ble for mediating knockdown of target genes. The targeted
`mRNA is than cleaved by activated RISC at a single site that
`is defined with regard to where the 5’-end of the antisense
`strand is bound to the mRNA target sequence [60, 61]. For
`RNAi-mediated mRNA cleavage and degradation to be suc-
`
`cessful, the double helix of the antisense-target mRNA du-
`plex must be in the A-form [62]. On the basis of these crite-
`ria, an effective siRNA has high stability (high content of G
`or C) at the 5’-terminus on the sense strand, in order to block
`incorporation of sense strand into RISC, lower stability at the
`5’-terminus of antisense (AU-richness) to promote incorpo-
`ration of antisense strand into RISC, and at the cleavage site
`(U at position 10 of sense strand), to help RISC-AS-
`mediated cleavage of mRNA and the RISC-AS-complex
`release. Effective gene silencing by RNAi machinery re-
`quires complete understanding of the elements that influence
`siRNA functioanality and specificity. These include, as well
`as only just described structural and sequence features re-
`quired, sequence space restrictions that define the boundaries
`of siRNA targeting. The sequence space is defined as the
`region of a gene that can be targeted for effective gene
`knockdown. Actually, targets are limited to regions of the
`gene that are transcribed: the 5’ and 3’ UTRs (untraslated
`regions) regions within 75 bases of the start codon and se-
`quences with > 50% C+G content and the ORF (open read-
`ing frame). Many computer programs are available for iden-
`tifying the optimal target sequences for a given gene [63,
`64]. The abundance of 3’UTR seed complements in off-
`target gene [65] suggested that, in the future, bioinformatic
`techniques that minimize off-target effects may be a standard
`component in siRNA design. There are multiple types of
`chemical modifications that are introduced into siRNAs in
`order to enhance thermodynamic and nuclease stability, to
`increase the half-life of the siRNA duplexes in vivo, to im-
`prove the biodistribution and pharmacokinetic properties of
`siRNAs, to target small RNAs to certain cell types and to
`improve the potency of siRNAs in terms of target binding
`affinity, and finally to reduce off-target effects. The synthe-
`sis and the properties of siRNAs bearing phosphodiester,
`carbohydrate and nucleobase modifications have been revi-
`wed extensively [66, 67]. Consequently, this review particu-
`larly covers the recent advances in this area that have been
`published over the past recent years, with particular attention
`on modified structure, architecture and conjugated siRNAs.(cid:1)
`
`CLASSICAL AND NOVEL CHEMICAL MODIFICA-
`TIONS OF siRNAs
`
` The current and most popular approach in the synthesis
`of oligoribonucleotides is based on phosphoramidite chemis-
`try which was originally developed for the synthesis of DNA
`oligonucleotides by Beaucage and Caruters in the early 1980
`[68]. Most siRNAs used in research today are made by
`chemical synthesis using phosphoramidite building blocks as
`single-stranded form. This approach permits incorporation of
`a wide variety of modifications into the siRNAs. A rational
`design of effective chemically modified siRNA must con-
`sider as general principle that the two strands of a siRNA
`function differently and as pratical hint that the nucleotides
`are different according to positions and nature. It has been
`recently demonstrated that novel sense and/or antisense
`strand modifications can greatly enhance siRNA specificity
`targeting, distinct and separate the events in interference
`cycle [69, 70].
`
`In this section, we will briefly review the most significant
`
`siRNA modifications described in literature, drawing atten-
`tion to those that have improved siRNA performance. We
`
`

`

`Recent Progress in Chemically Modified siRNAs
`
`Mini-Reviews in Medicinal Chemistry, 2010, Vol. 10, No. 7 581
`
`will point out the useful and universal modifications as well
`as the most innovative modifications to move siRNA toward
`the clinic.
`
`Base Modifications
`
` Base-modified siRNAs (Fig. 1) have been investigated so
`far to a very limited extent [71, 72] despite the central in-
`volvement in target recognition of nucleobases. The report
`by Parrish et al. describe induction of RNAi in C. elegans by
`dsRNAs containing 4-thiouridine, 5-bromo-, 5-iodo-, 5-(3-
`aminoallyl)-uridine, or inosine [71]. While these experiments
`did not demonstrate any remarkable differences in silencing
`activity regardless of the introduced modifications, the work
`by Chiu and Rana [72] describes reduced silencing activity
`of duplexes containing 5-bromouridine, 5-iodouridine, 2,6-
`diaminopurine, and N-3-methyl-uridine. The patent literature
`also claims several base-modified nucleosides as compo-
`nents of small interfering nucleic acids, albeit no details are
`provided with regard to properties of such modified siRNAs
`[73]. In other studies [74], Sipa et al. evaluated the thermo-
`dynamic stability and gene-silencing activity of base-
`modified siRNAs containing three modified nucleosides: 2-
`thiouridine (s2U), pseudouridine ((cid:1)), and dihydrouridine (D).
`They demonstrated that these three naturally occurring modi-
`fied nucleosides, when introduced into a siRNA duplex,
`modulate its silencing potency. The extent of this effect de-
`pends on the modification position and is most advantageous
`when the s2U, (cid:1), and D nucleosides participate in an en-
`hancement of the siRNA duplex asymmetry. More recently,
`Kool at al. have evaluated steric and stability effects on
`RNAi activity by use of siRNAs modified with propynyl and
`methyl functionalities at the C-5 position of pyrimidine nu-
`
`cleobases [75]. Their results suggest that at the 5’-half of the
`guide RNA, large increases in the size of the functionality at
`the position 5 of pyrimidine nucleobases can be detrimental
`to RNAi activity. Despite the strong stabilizing effects of the
`5-propynyl modification, this bulky substitution caused
`negative effects in RNAi activity, probably due to disruption
`of interactions in the major groove of the active RISC com-
`plex. However, the smaller 5-methyl substitution did not
`adversely affect gene silencing activity. Surprisingly, some
`atypical base structures have been used in siRNA [76].
`SiRNA duplexes containing the 2,4-difluorotoluyl ribonu-
`cleoside (rF) were synthesized to evaluate the effect of non-
`canonical nucleoside mimetics on RNA interference. 5´-
`Modification of the guide strand with rF did not alter silenc-
`ing relative to unmodified control. Internal uridine to rF sub-
`stitutions were well-tolerated and thermal melting analysis
`showed that the base pair between rF and adenosine (A) was
`destabilizing relative to a uridine-adenosine pair, although it
`was slightly less destabilizing than other mismatches. siR-
`NAs with the rF modification effectively silenced gene ex-
`pression and offered improved nuclease resistance in serum.
`Base modifications can also help to reduce immune activa-
`tion: modification with pseudouracil or 2-thiouracil prevents
`the RIG-I-mediated
`immunostimulation due
`to a 5’-
`triphosphate [40], and 5-methyl-C, N6-methyl-A pseu-
`douridine prevents recognition of RNA by TLR3, TLR7 and
`TLR8 [77].
`
`Sugar Modifications
`
` The siRNA modifications on the sugar moiety are the
`most widely described (Fig. 2). The milestone study report-
`ing the pioneering analysis of chemical modified siRNA
`
`F
`
`R
`
`rF
`
`N
`
`N
`
`R
`
`F
`
`NH2
`
`N
`
`Br
`
`N
`
`NH2
`
`O
`
`N
`
`R
`
`I
`
`NH
`
`O
`
`O
`
`N
`
`R
`
`NH
`
`O
`
`2,6-DAP
`
`5-BrU
`
`5-IU
`
`O
`
`O
`
`HN
`
`HN
`
`HN
`
`NH
`
`~) ~) 4
`Y~ Y~ Y~
`
`O
`
`R
`
`(cid:1)
`
`O
`
`NH
`
`O
`
`NH2
`
`N
`
`N
`
`I
`
`R
`
`O
`
`O
`
`N
`
`R
`
`s2U
`
`S
`
`O
`
`N
`
`R
`
`D
`
`O
`
`NH
`
`O
`
`N
`
`I
`
`R
`
`N
`
`I
`
`R
`
`pU
`
`mU
`
`mC
`
`
`
`Fig. (1). Nucleobase modifications in siRNA. rF: 2,4-difluorotoluyl ribonucleoside; 2,6-DAP: 2,6-diaminopurine; 5-BrU: 5-bromouridine; 5-
`IU: 5-Iodouridine; s2U: 2-thiouridine; D: dihydrouridine; (cid:1): pseudouridine; pU: 5-propynyluridine; mU: 5-methyluridine; mC: 5-
`methylcitydine
`
`

`

`582 Mini-Reviews in Medicinal Chemistry, 2010, Vol. 10, No. 7
`
`Gaglione and Messere
`
`function [72] showed that while A-form helix is required for
`the mechanism of RNAi, the 2’-OH function was not an es-
`sential requirement for interfering activity. This study
`strongly indicates that RNAi machinery does not require the
`2’-OH for recognition of siRNAs and catalytic ribonuclease
`activity of RISC does not involve the 2’-hydroxyl group of
`guide antisense of RNA. Therefore, the 2’ position of ribose
`has been heavily modified. Modification of the 2’ position of
`the ribose can increase Tm of duplex and confers varying
`degrees of nuclease resistance. It may also provide protection
`from immune activation. Briefly, we list here the most ex-
`tensively studied 2’ modifications in siRNA effective mole-
`cules. 2’-fluoro (2’-F) was among the first modifications that
`were independently tested by several groups and they result
`well tolerated in siRNA applications [71, 72, 78-80]. 2’-O-
`methyl (2’O-Me) was extensively tested in siRNA by differ-
`ent groups since 2003 [72, 79-87]. Partial modification of
`siRNA strands with 2’O-Me (less than 8 nt) led to improved
`performance of siRNA in cell [86], but siRNAs completely
`made of 2’O-Me lost interference activity [72]. Position-
`specific 2’-O-methylation of siRNAs reduces “off target”
`transcript silencing [69]. The sharp position dependence of
`2’-O-Me modification contrasts with the broader position
`dependence of base-pair within the seed region, suggesting a
`role for position 2 of the guide strand distinct from its effects
`on pairing to target transcripts. Besides aforementioned sta-
`bility, 2’-O-Me siRNAs exhibit increased potency [88] and
`reduced immunostimulatory activity [41, 89], with the ex-
`ception 2’-O-Me cytidine [90]. Introduction of 2’-O-Me
`uridine or guanosine into one strand of the siRNA duplex
`generates noninflammatory siRNAs. 2’-O-Me siRNAs, con-
`taining less than 20% modified nucleosides and targeting
`apolipoprotein B (apoB), can mediate potent silencing of its
`target mRNA and cause significant decreases in serum apoB
`and cholesterol. This is achieved at therapeutical viable
`siRNA doses without cytokine induction, toxicity or off tar-
`get effects [90]. It was been showed that 2’-O-Me RNAs act
`as potent antagonists of immunostimulatory RNA and they
`are able significantly to reduce both interferon-(cid:1), and inter-
`leukin-6 induction by small-molecule TLR7 agonist loxorib-
`ine in human peripheral blood mononuclear cells, murine
`Flt3L dendritic cells and in vivo mice [89]. Further, 2’-O-
`methylation selectively protects the particularly vulnerable
`5’-end of the guide strand against exonucleases in human
`blood serum [91]. Specific chemical modification thus re-
`solves the asymmetric degradation of the guide and sense
`strands, which is inherent to the thermodynamic asymmetry
`of the siRNA termini as required for proper utilization of the
`guide strand in RNAi pathway. The 2’-O-(2-methoxyethyl)
`residues (2’-O-MOE) can be incorporated into siRNAs much
`like 2’-O-Me or 2’-F. The 2’-MOE modification in the an-
`tisense strand resulted in less active siRNA constructs re-
`gardless of placement position in the construct. The incorpo-
`ration of modified residues in the sense strand did not show a
`strong positional preference [85]. This modification is not
`generally available for use but a successful gene target in
`vivo is reported [92]. Likewise, 2’-O-allyl modification is
`well tolerated in the 3’-overhangs but not at most positions
`in siRNA duplexes [87]. Another class of 2’ modification is
`2’-deoxy-2’-fluoro-(cid:2)-D-arabino nucleic acids
`(FANA).
`FANA nucleotides have the stereochemistry at the 2’ posi-
`
`tion inverted relative to that found in 2’-O-Me and 2’-F
`RNA. siRNAs that have completely FANA-substituted sense
`strands are 4-fold more potent than unmodified siRNAs and
`have a longer half-life in serum. The antisense strand is less
`tolerant of the FANA modification [93, 94]. DNA itself rep-
`resents a further modification in which there is not the elec-
`tronegative OH-group in 2’ position. The use of DNA in 3’-
`overhangs of synthetic siRNAs is well known [27]. Substitu-
`tion with double stranded DNA in the 8-bp region at the
`5’end of the guide strand gives active duplexes with reduced
`off-target effects [95]. Interestingly, 2’deoxy bases have re-
`cently been reported to also block immune detection, particu-
`larly T or dU bases [96]. Locked nucleic acids is a family of
`conformationally locked nucleotide analogues containing a
`methylene bridge between the 2’ and 4’ carbons of the ribose
`ring [97]. This linkage constrains the ribose ring, “locking” it
`into the 3’-endo conformation close to that formed by RNA
`after hybridization. The incorporation of LNA in siRNAs
`enhances serum half-life substantially and a few LNA units
`at the 5’-end of the sense strand improves thermodynamic
`bias, reduces immunostimulatory and off-target effects [39,
`98]. Particular LNA based siRNAs were designed and de-
`veloped against the highly structured 5’-UTR of cox-
`sackievirus B3 (CVB-3) [99]. The best siLNA improved
`viability of infected cells by 92% and exerted good antiviral
`activity in plaque reduction assays. 2’-O-(2,4-dinitrophenyl)
`RNAs were among the 2’modification that have been more
`recently tested in the context of RNAi. DPN-ssRNA and
`DPN-siRNA outperform previous generation of antisense
`and unmodified siRNAs in gene silencing potency, stability,
`hybridization affinity and delivery maintaining specific bind-
`ing properties. Poly-2’-O-(2,4-dinitrophenyl)-oligoribonucl-
`eotide (DNP-RNA) represents a promising new gene silenc-
`ing platform. DNA-RNAs with a DNP nucleotide molar ratio
`of about 0.7 can spontaneously cross viral envelopes and
`mammalian cell membranes. DNP-RNAs are resistant to
`degradation by ribonucleases, including RNases A, B, H, S,
`T 1 and 2 and phosphodiesterase I and II [100] and hybridize
`with their complementary RNA. Interestingly, 2’-O-DNP
`modified siRNA and its single stranded counter part have
`shown similar silencing activities [101-103]. The ring oxy-
`gen has also been modified: 4’-thio modified nucleosides
`have a sulfur atom at the 4 position of the ribose ring. Ho-
`shika et al. report that 4’-thio RNAs form a thermally stable
`duplex with the complementary RNA and show high nucle-
`ase resistance, despite the 2’-OH groups. In addition, struc-
`tural analysis by CD spectra
`indicates
`that
`the 4’-
`thioRNA:RNA duplex adopts an A-form conformation as
`does the natural RNA duplex. 4’-S-RNA is very well toler-
`ated near the termini of siRNA duplexes [104-106]. Modifi-
`cations at the sense-strand preserve RNAi activity of modi-
`fied siRNA, except
`for
`full modification with 4’-
`thioribonucleoside and the activity of siRNAs modified at
`the antisense-strand was dependent on the position and the
`number of modifications with 4’-thioribonucleosides [104].
`For example, the 5’-end of the antisense strand could be
`modified with a few 4’S-RNA inserts, without significant
`loss of potency [105]. Furthermore, Dande et al. describe
`that significant improvements in siRNA activity and plasma
`stability were achieved by judicious combination of 4’-
`thioribose with 2’-O-Me and 2’-O-MOE modifications. It is
`
`

`

`Recent Progress in Chemically Modified siRNAs
`
`Mini-Reviews in Medicinal Chemistry, 2010, Vol. 10, No. 7 583
`
`O
`
`/1-
`
`B
`
`B
`
`O
`
`O
`
`O
`
`O
`
`)-,..
`
`B
`
`O
`
`/1-
`
`O
`
`B
`
`O
`
`O
`
`O
`
`2'-MOE
`
`OCH3
`
`O
`
`B
`
`O
`
`O
`
`2'-OMe
`
`~ I
`
`F
`
`B
`
`O
`
`O
`
`OH
`
`O
`
`F
`
`O
`
`B
`
`O
`
`2'-F
`
`O
`
`O
`
`)-,..
`
`B
`
`2'-OH
`
`O
`
`/1-
`
`O
`
`O
`
`O
`
`O
`
`O
`
`O
`
`1, c l,c>-:c
`L 1\)-,.. I, C
`I" j__
`>-
`l,c
`I,{ l,c
`I, C I, C
`
`B
`
`S
`
`O
`
`OH
`
`4'-THIO
`
`;,..,,,
`
`O
`
`B
`
`O
`
`I-((
`
`O
`
`O
`
`LNA
`
`~
`
`O
`
`2'-FANA
`
`O
`
`2'-deoxy
`
`O2N
`2'-DNP
`
`;!'
`
`2'-O-allyl
`
`)-,.. 0
`
`B
`
`O
`
`O
`
`NH2
`
`2'-amino
`
`/1-
`
`O
`
`B
`
`O
`
`NO2
`
`O
`
`O
`
`OH
`
`UNA
`
`B
`
`
`
`A
`
`O
`
`1-ztr
`
`O
`
`O
`
`ENA
`
`Fig. (2). Sugar modifications in siRNA. 2’-F: 2’-fluoroRNA; 2’-OMe: 2’-O-Methyl RNA; 2’-MOE: 2’-O-(2-methoxyethyl)RNA; 2’-O-allyl:
`2’-O-allyl RNA; 2’-DNP: 2’-O-(2,4-dinitrophenyl) RNA; 2’-FANA: 2’-deoxy-2’-fluoro-(cid:2)-D-arabino nucleic acids; 2’-deoxy: 2’-
`deoxyribonucleotide; LNA: locked nucleic acid; 4’-Thio: 4’-thioribonucleoside; UNA: unlocked nucleic acid; 2’-amino: 2’-amino RNA;
`ENA: ethylene-bridge nucleic acid.
`
`also possible to combine 4(cid:1)-thio substitution with FANA
`nucleotides, and 4(cid:1)-S-FANA–modified siRNAs have been
`shown to possess potencies for silencing in cell culture that
`were
`similar
`to unmodified
`siRNAs
`[95]. 2’,3’-
`Seconucleosid