Chemical and Structural Diversity of siRNA Molecules
`
`Current Topics in Medicinal Chemistry, 2006, 6, 913-925
`
`913
`
`Barbara Nawrot* and Katarzyna Sipa
`
`Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences,
`Sienkiewicza 112, 90-363 Lodz, Poland
`
`Abstract: Short interfering RNAs (siRNAs) are 21-23 nt long double-stranded oligoribonucleotides which in mammalian
`cells exhibit a potency for sequence-specific gene silencing via an RNA interference (RNAi) pathway. It has been already
`proven that exogenous, chemically synthesized siRNA molecules are effective inhibitors of gene expression and are
`widely applied for analysis of protein function and proteomics-based target identification. Moreover, since their discovery
`siRNA molecules have been implemented as potential candidates for therapeutic applications. Variously modified siRNA
`molecules containing sugar modifications (2’-OMe, -F, -O-allyl, -amino, orthoesters and LNA analogues), internucleotide
`phospodiester bond modifications (phosphorothioates, boranophosphates), base modifications (s2U) as well as 3’-terminal
`cholesterol-conjugated constructs were investigated as potential candidates for effective inhibition of gene expression.
`This chapter reviews an impact of chemical and structural modifications of siRNA molecules on their serum and thermal
`stability, cellular and in vivo activity, cellular uptake, biodistribution and cytotoxicity. Functional analysis of chemically
`modified siRNA molecules allows for better understanding of the mechanism of the RNA interference process as well as
`demonstrates immense efforts in optimizing in vivo potency of siRNA molecules for RNAi-based drug design.
`
`Keywords: RNA interference, small interfering RNA, gene silencing, siRNA, modified siRNA, chemical modification, RNA
`structure.
`
`INTRODUCTION
`
`An initial enthusiasm paid to antisense strategy deve-
`loped in 80-ties of past century using DNA oligonucleotides
`and their analogues has not fulfilled early expectations to
`date [1, 2]. Only one antisense oligo(nucleoside phos-
`phorothioate) has been approved till now as a drug against
`cytomegaloviral (CMV) infection in HIV-1 infected patients
`(Fomivirsen, Vitravene®, Isis Pharmaceuticals, 1998) [3].
`Another oligonucleotide, active as an aptamer of vascular
`endothelial growth factor (VEGF) was approved by FDA in
`2005 (Macugen®, Pfizer) for age-related macular degene-
`ration (AMD) therapy [4]. New hopes for sequence-specific
`control of gene expression emerged with the discovery of
`RNA interference (RNAi) mechanism. Shortly after Fire’s
`first report on RNAi in Caernohabditis elegans [5], some of
`its features were found common to other mechanisms of
`post-transcriptional gene silencing in many different species
`like plants [6], fungi [7] and insects [8]. In these mechanisms
`common is the function of short ribonucleic acids, so far
`much underestimated as less stable members of nucleic acids
`family.
`The post-transcriptional gene silencing is based on
`degradation of mRNA complementary to exogenous double
`stranded RNA. Three years after discovery of the phenome-
`non of RNA interference, the process was intentionally
`induced in mammalian cells [9]. That was possible due to the
`use of synthetic short interfering RNA (siRNA) duplexes
`which do not induce antiviral defense system in eukaryotic
`
`*Address correspondence to this author at the Department of Bioorganic
`Chemistry, Centre of Molecular and Macromolecular Studies, Polish
`Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland; Tel: 0048-
`42-681 69 70; Fax: 0048-42-681 54 83; E-mail: bnawrot@bio.cbmm.lodz.pl
`
`cells. In spite of the fact that antiviral defense system
`recognizes long double stranded RNA (> 30 bp) as a signal
`for apoptosis and induces interferon response [10], scientists
`soon started to consider siRNA molecules as the most
`promising tool for gene silencing in mammalian cells as well
`as in higher organisms.
`Although siRNA molecules suffer from their low stabi-
`lity in vivo, insufficient cellular uptake and induced off-
`target effects, they seem to be more useful than antisense
`oligonucleotides for analysis of function of newly discovered
`genes, and in silencing of genes to combat certain diseases.
`Nonetheless, significant progress was made to overcome
`these difficulties, which capitalized on knowledge gained
`during attempts at chemical modification of antisense
`oligonucleotides. These achievements made the siRNA
`molecules more potent and more useful, albeit yet much
`unpredictable. Thus, application of the siRNA molecules
`requires further studies, also in respect to new chemical
`modifications of these tools improving their in-serum and
`thermal stability, intracellular and in vivo activity, cellular
`uptake, biodistribution and low cytotoxicity.
`
`RNAi IN MAMMALIAN CELLS
`
`In general, an RNAi phenomenon involves three major
`steps [see Fig. (1)]. Within the first step, the Dicer nuclease
`specifically hydrolyzes long double stranded RNA into
`siRNAs. The one strand of the resulting siRNA duplex is
`incorporated into a protein complex, called RISC (RNA
`Induced Silencing Complex). Activated in this way RISC
`hydrolyzes phosphodiester bond in the target mRNA in a
`region complementary to the guide strand of siRNA [11, 12].
`The steps of Dicer-promoted formation of siRNA and
`unwinding of siRNA duplex just before incorporation of one
`strand into the RISC [13], both depend on ATP. However,
`
` 1568-0266/06 $50.00+.00
`
`© 2006 Bentham Science Publishers Ltd.
`
`Alnylam Exh. 1033
`
`

`

`914 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 9
`
`Nawrot and Sipa
`
`dsRNA
`
`shRNA
`
`I
`
`II
`
`cap
`
`III
`
`t siRNA generation
`~~ siRNA
`t Complex activation
`
`t sequence-specific hydrolysis
`
`Gene silencing
`
`Fig. (1). Mechanism of gene silencing by induction of RNA interference. Step I: long double stranded RNA is recognized by nuclease Dicer
`and cleaved into 21-23 nt short interfering RNAs (siRNAs). Step II: nuclease-helicase complex RISC (RNA Induced Silencing Complex) is
`activated by incorporation of the antisense strand of siRNA duplex. Step III: activated RISC guided by the antisense strand of siRNA
`recognizes complementary strand of mRNA and catalyzes cleavage of the target sequence. The cleaved mRNA is further degraded by
`cellular nucleases. Degradation of mRNA leads to silencing of the coded gene.
`
`neither the exact mechanism nor the proteins involved in
`these processes are so far well established. The RISC-
`promoted hydrolysis of mRNA does not consume ATP and
`takes place only in the presence of Mg2+ cations to yield the
`5’-end phosphorylated product [14, 15]. The cleaved mRNA,
`deprived of the natural protection at the cleavage site, is
`immediately recognized by the cellular nucleases and
`promptly degraded. It is assumed that the activated RISC
`may hydrolyze several molecules of the target mRNA [13,
`16, 17], while bound siRNA chain acts as the guide strand
`
`recognizing the target sequence within a coding region of
`mRNA [15].
`Another mechanism resulting in steric blocking of
`protein biosynthesis is switched on when the RNA strand
`present in RISC complex is not fully complementary to the
`3’-untranslated region of mRNA (3’-UTR) [18-20]. In this
`case, the RNA strand incorporated into RISC comes from so
`called micro RNA family of short RNA molecules
`possessing a hairpin structure. The principal difference
`between RNAi and micro RNA mechanisms is the origin of
`
`

`

`Chemical and Structural Diversity of siRNA Molecules
`
`Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 9 915
`
`precursors, which for miRNA are encoded in the genome.
`Short RNAs may also regulate gene expression during the
`transcription as they are involved in condensation of
`heterochromatin [21]. In all these processes similar proteins
`(belonging to the Argonaute family) are involved. However,
`natural mechanisms of control of the gene expression, in
`which short nucleic acids play a main role, are not fully
`understood yet and it is difficult to assess without doubts
`possible relations and similarities.
`The process of RNA interference in mammalian cells can
`be induced with the omitting of the first step [Fig. (1)].
`Sequence specific gene silencing in mammalian cells was
`achieved by delivery to the cells of either ready to use
`siRNAs [9, 12] or their precursors, short hairpin RNAs
`----
`(shRNAs), which are the substrates for the Dicer nuclease
`[22, 23]. The siRNA and shRNAs molecules can be genera-
`ted endogenously or synthesized by chemical either enzy-
`matic approach. Among different vectors encoding shRNA
`under the control of the RNA polymerase III (Pol III)
`promoters the viral vectors including lentiviral and adeno-
`associated vectors seem to be the most promising [24, 25].
`Unfortunately, these strategies suffer from possible severe
`side effects like restoration of the viral virulence. Another
`approach was explored based on the synthesis of the total
`pool of so called esiRNAs, which could be generated in vitro
`by RNase III-assisted cleavage of long double stranded RNA
`complementary to the target gene [26]. The effectiveness of
`prepared on such way oligomers is usually high, but
`selection of sufficiently specific target sequence is difficult.
`Historically first and still widely used method of preparation
`of siRNA or shRNA is the chemical synthesis, which offers
`full control of the structure of the product in respect to the
`sequence and purposely introduced chemical modifications.
`Moreover, synthetic siRNA molecules, possessing chemical
`modifications in defined regions of the duplex, enabled to
`gain information on the mechanism of binding of the siRNA
`strand into RISC [27, 28].
`The aim of this account is to present the state of the art
`concerning the knowledge on synthetic siRNAs, their desired
`features, selection of active target sequences, and new
`options coming from chemical modifications in duplexes.
`
`STRUCTURAL DIVERSITY OF siRNA DUPLEXES
`
`siRNA molecules were identified in cellular extracts
`from Drosophila melanogaster embryos transfected with
`dsRNA [12]. The majority of isolated short RNAs were 20-
`23-bp duplexes. Further experiments demonstrated that the
`most potent siRNA molecules were 19-bp RNA duplexes
`with 2-nt overhangs at their 3’-ends [29] [Fig. (2A)]. These
`two overhanging nucleotides could be replaced with DNA
`units, preferably
`thymidines [29]. Subsequent studies
`showed that such modified duplexes were less active at
`lower concentrations [30]. The family of siRNA duplexes
`has been extended recently with blunt-ended constructs [31,
`32]. Also, increased activity was found for fork siRNA
`duplexes possessing not
`fully complementary
`(two
`mismatches) blunt ends [33]. The presence of the two
`unpaired nucleotides might be beneficial for the RNA-
`protein interaction due to the dinucleotide-binding pocket
`identified at PAZ domain of Argonaute protein [34-36].
`However, since the pattern of interactions within RISC is not
`
`well understood, above experimental observations of high
`activity of blunt-ended siRNAs must be interpreted with
`caution.
`The siRNA duplexes of various lengths were tested for
`their ability to induce RNA interference. Recently, it was
`demonstrated that 27-bp RNA duplexes were the substrates
`for Dicer nuclease and were very active in cellular system
`[37, 38]. Also shorter 19-bp blunt siRNA duplexes exhibited
`remarkable silencing activity, while 17-bp constructs were
`non-effective, even in the presence of additional target-
`specific overhangs [31]. Interestingly, a 19-bp long siRNA
`duplex not fully complementary to the terminal positions of
`the target sequence (with up to four mismatches) still
`mediated efficient gene silencing, suggesting that the base
`pairing with the target mRNA is not the most decisive factor
`determining the minimal length of functional siRNA.
`Important information was gained from chemical modi-
`fications of different regions of the siRNA duplex. It was
`found that the 3’-ends could be freely modified, while the 5’-
`ends, especially the 5’-end of the antisense strand, does not
`tolerate any modification [Fig. (2B)]. The recently published
`structure of the PIWI domain of RNA-binding Argonaute
`protein confirmed the importance of the 5’-phosphate group
`for the interaction within RISC [39, 40].
`The effects caused by point mutations differed depending
`on the site of modification. Modifications within the sense
`strand did not affect the activity, even if several mismatches
`were introduced. Single modifications close to the ends of
`the antisense strand did not reduce the activity significantly
`on the contrary to those introduced in the middle [41, 42].
`These observations are in accordance with the in vitro
`analysis showing that the cleavage of target mRNA takes
`place at the phosphate opposite of nucleotides 10 and 11 of
`the guide strand of siRNA (starting from the 5’-end
`phosphate moiety) [29]. Notably, the synthetic siRNAs are
`effectively phosphorylated at the 5’-end by intracellular
`kinases [16, 17, 43].
`One has to notice that different siRNA duplexes,
`designed according to the mentioned above rules (duplex
`length, presence of 3’-overhangs, sequence complementarity
`in the centre and presence of the phosphate group at the 5’-
`terminus of the antisense strand), act differently in silencing
`of the same target gene. It is well known that even small
`siRNA sequence shift significantly alters silencing activity
`[44]. Such observations support conclusion that siRNA
`sequence itself, and not a local secondary structure of the
`target mRNA molecule, is crucial for duplex potency.
`However,
`there are some reports supporting opposite
`dependence [45-47]. Important information about siRNA
`activity determinants was obtained from studies on the
`mechanism of incorporation of the RNA strand into RISC. It
`was found that the unwinding of siRNA duplex is not
`symmetrical and the strand finally incorporated is that with
`weaker hybridization at its 5’-end [27, 28]. Decisive
`influence on activity of siRNA is attributed to the nucleotide
`composition – preferably with more A and U units close to
`the 5’-end of the strand dedicated as a guide strand.
`Moreover, sequences able to form structures of higher order
`should be avoided. These rules were used for algorithms of
`web browsers of potentially active sequences of siRNAs,
`
`

`

`916 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 9
`A
`
`Wild type siRNA
`
`19-bp siRNA duplex
`with 3'-1T overhangs
`
`Blunt-ended siRNA
`
`Fork siRNA
`(non-complementary
`al 5 '-end of the
`anti sense strand)
`
`Nawrot and Sipa
`
`3'
`
`5'
`
`3,-«WJIID I i I I I I I I t Ctwl;-
`
`~ 3 •
`
`5 '
`3'-t::~::!:::!=:::!:::~:::!=:::'.=::::::~==~=>5 '
`
`B
`
`,--(cid:144)
`
`S'-
`
`sense strand
`
`0-3•
`121 111111 I 8 I 1111111 I ~.!.
`
`antlsense strand
`
`(cid:144)
`@
`
`Variable groups
`
`phosphate
`
`-
`Strand regions accessible for modifications
`Fig. (2). (A) Structural diversity of active siRNA duplexes. (B) Possible chemical modification sites in siRNA duplex. Modifications located
`at 3’-ends of the sense and antisense strands as well as at 5’-end of the sense strand are accepted to maintain siRNA silencing activity.
`Phosphate group at the 5’ terminus of the antisense strand is required for functional siRNA duplex. Modifications at this site are allowed only
`when they are attached to the terminal phosphate group. Introduction of modifications at the centre of the antisense strand (opposite to
`scissile phosphate, marked with an ellipse) leads to a loss of the silencing activity of the siRNA duplex.
`
`offered by vendors synthesizing siRNAs (e.g. Dharmacon,
`Ambion) [48, 49] or by independent web browsers [50, 51].
`One of such tools is Deqor program [52] searching for longer
`sequences suitable for generation of esiRNA molecules.
`There is no algorithm assuring high activity of the selected
`siRNA. Despite of the careful selection of the target
`sequence and
`its specificity, siRNAs may affect
`the
`expression of many other genes in a cell, causing so called
`“off-target effects” [53-58]. This fact emphasizes
`the
`importance of detailed search for intracellular processes in
`which siRNA-like molecules are involved. There is also
`some hope that observed off-target effects, which are
`concentration-dependent, can be limited by application of
`new modifications making the siRNA probes more effective.
`
`CHEMICAL MODIFICATIONS OF siRNA MOLE-
`CULES
`
`RNA interference induced by synthetic siRNA molecules
`is a transient process. In mammalian cells re-expression of
`
`the target gene usually occurs after few days [59]. Therefore,
`an improvement of cellular stability of the siRNA molecules
`is of interest to design in vivo effective gene silencers.
`Although variety of chemical modifications of RNA strands
`are intended mostly to enhance activity and specificity, an
`increased stability of siRNA in living systems is highly
`appreciated. Up to now several review papers summarizing
`the influence of the chemical modifications within siRNA
`strands on the in vitro and in vivo activity and specificity of
`siRNA molecules were published [60-63].
`
`Chemical Modifications Improving Resistance of siRNA
`Molecules Towards Nucleases
`
`Natural nucleic acids are easily degraded by intracellular
`nucleases. This process may be slowed down, or even
`terminated, by chemical modification of oligonucleotide
`strands of siRNA duplexes. Chemical modifications of
`siRNA molecules fall into several categories, including
`modifications of the internucleotide phosphodiester bond
`
`

`

`Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 9 917
`
`complementary RNA strands [70]. The stability of the PS-
`siRNA duplexes in serum is very high (up to 72 h of
`incubation) in comparison to PO-siRNA duplexes [71-73]. In
`general, phosphorothioate modifications of siRNA duplexes
`do not interfere with their silencing activity [41, 44, 70, 74],
`although it was also shown that PS-siRNAs alter silencing
`from medium to high level [72]. In experiments reported by
`Harborth et al. [44], phosphorothioate bonds introduced at
`the 5’- and 3’-ends of both, antisense and sense strands of
`siRNA, or at juxtaposed position of the oligonucleotide
`chains did maintain strong efficiency for silencing of lamin
`A/C. On the contrary, Chiu and Rana reported that the
`presence of the phosphorothioate modifications in the sense
`strand (which is not involved in the mRNA hydrolysis)
`reduced the activity of siRNA by ca. 40 % [71]. Numerous
`reports presenting applications of various PS-siRNA
`molecules for effective silencing of the target genes are
`summarized in Table 1. Some of them describe PS-siRNAs
`with increased toxicity, especially those containing more
`than half of the internucleotide bonds modified [41, 44, 72].
`This unwanted effect was avoided by the use of the siRNA in
`lower concentrations [72]. An increased nuclease resistance
`of PS-modified siRNAs is beneficial for their pharma-
`cokinetic properties [72]. The in vivo studies showed that the
`siRNA duplexes consisting of antisense PO- and sense PS-
`RNA strands were present in blood of animals at higher
`concentration than their unmodified PO/PO counterparts [73].
`Boranophosphate analogues of siRNA obtained enzy-
`matically using stereospecific T7 RNA polymerase represent
`another interesting tool for potential therapeutic application
`[75]. These compounds obtained as stereodefined diastereo-
`isomers of SP configuration at the P-chiral center (all-SP
`oligomers) were more lypophilic (yet carrying a negative
`
`Chemical and Structural Diversity of siRNA Molecules
`
`I ~/
`
`O
`
`-X
`
`B
`
`O
`
`X = O RNA
`X = S PS-RNA
`X = BH3 PB-RNA
`
`B
`
`OH
`
`O
`
`P
`
`O
`
`OH
`
`O
`
`O
`
`O
`
`""'-:(f 11--1.i_
`
`Fig. (3). Structures of RNA (X=O), phosphorothioate RNA (X=S)
`and boranophosphate RNA (X=BH3) fragments.
`
`[Fig. (3)], ribose ring (substituent at 2’-carbon atom, LNA,
`ENA, see Fig. (4)], nucleobases [Fig. (5)] as well as those
`with different “tags” attached at either 5’- or 3’-ends [Fig.
`(6)].
`
`Modifications of the Phosphodiester Bond
`
`Replacement of sulfur atom for one of the non-bridging
`oxygen atoms in phosphate internucleotide bond is widely
`used to protect exogenous oligonucleotides against nucleo-
`lytic hydrolysis [64-66] [Fig. (3)]. Such strategy was widely
`used to protect antisense DNA oligonucleotides tested for
`therapeutic applications [67-69]. The diastereomeric mixture
`of the chemically synthesized phosphorothioate oligonucleo-
`tides forms thermodynamically less stable duplexes with
`
`',_<i~-m
`
`B
`
`O
`
`O
`
`/
`
`O
`
`O
`
`P
`
`\
`
`O
`
`-
`
`O
`
`O
`
`O
`
`B
`
`O
`
`O
`
`',_
`
`B
`
`B
`
`O
`
`O
`
`,~,,m_
`
`O
`
`O
`
`P
`
`O
`
`O
`
`O
`
`-
`
`\
`
`O
`
`O
`
`/
`
`O
`
`'r{''xi
`
`R
`
`O
`
`B
`
`O
`
`O
`
`O
`
`P
`
`-
`O
`
`\
`
`O
`
`R
`
`B
`
`O
`
`/
`
`O
`
`LNA ENA
`
`R = OH RNA
`R = O-CH3 2'-OMe RNA
`R = O-CH2CH2OCH3 2'-OMOE RNA
`R = O-CH(CH2CH2 OH)2 2'-orthoes ter RNA
`R = H DNA
`R = F 2'-F RNA
`R = 2,4-dinitrophenyl 2'-DNP RNA
`R = NH2 2'-NH2 RNA
`R = O-CH2-CH=CH2 2'-allyl RNA
`
`Fig. (4). Structures of RNA analogues possessing modifications in the 2’-position of the ribose ring. B constitutes nucleobase (uracil,
`cytosine, adenine or guanine) residue.
`
`

`

`918 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 9
`
`Nawrot and Sipa
`
`O
`
`N
`
`R
`
`NH
`
`S
`
`O
`
`HN
`
`NH
`
`O
`
`R
`
`S
`
`N
`
`R
`
`NH
`
`O
`
`{,(
`
`CH3
`
`N
`
`O
`
`O
`
`N
`I
`R
`
`2-thiouridine
` (s2U)
`
`pseudouridine
`(Y
`
`)
`
`4-thiouridine
` (s 4U)
`
`N-3-Me-uridine
` (m3 U)
`
`NH2
`
`N
`
`N
`
`NH2
`
`N N
`
`R
`
`<:6
`
`O
`
`N
`
`NH
`
`N N
`
`I
`R
`
`X
`
`O
`
`N
`R
`
`NH
`
`O
`
`X = Br 5-bromouridine (br5U)
`X = I 5-iodouridine (i 5U)
`X = CH=CHCH2NH2
` 5-(3-aminoallyl)-uridine
`Fig. (5). Structures of base-modified ribonucleosides used for siRNA structure/activity studies. R constitutes b
`
`inosine
` (I)
`
`2,6-diaminopurine
` ribonucleoside
`
`,D-ribofuranoside residue.
`
`H
`
`H
`
`H
`
`H
`
`H
`
`O
`
`NH
`
`-~
`
`RNA O
`
`choles teryl-RNA
`
`,,,,,,
`
`Me
`
`N
`
`M e
`
`N
`
`N
`
`N N
`
`RNA
`
`O
`
`O
`
`NH
`
`OH
`
`O
`
`NH2
`
`OMe
`
`5'-puromycin-RNA
`
`OR
`
`NA
`
`I =-I
`
`O
`
`P O-
`
`O
`
`O RNA
`
`O
`
`4
`
`O-
`
`P
`
`O
`
`4
`
`O
`
`OH
`
`/
`RNA
`
`125 I-labeled tyrosine residue
`attached to RNA chain
`
`NH
`
`O
`
`-
`
`O2C
`
`O
`
`O
`
`OH
`
`I -0
`
`I
`
`OH
`
`CH2
`
`NH2
`CH
`=
`CO
`NH2
`( I )
`CH2
`
`<D
`--q
`~ ~
`
`5'-fluorescein-RNA
`
`3'-fluores cein-RNA
`
`-
`O
`CO2
`Fig. (6). Structures of RNA conjugates with 3’-attached cholesteryl residue, radioactively labeled tyrosine residue or with fluorescein attached
`at 3’- or 5’-terminus of the sense strand.
`
`O
`
`OH
`
`NH
`
`O
`
`5
`
`HN
`
`

`

`Chemical and Structural Diversity of siRNA Molecules
`
`Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 9 919
`
`Table 1.
`
`Selected Examples of Silencing Activity of PS-Modified siRNA Molecules
`
`Duplex ID
`
`Site of modification (underlined)
` 5’fi
` 3’ (sense)
` 3’‹
` 5’ (antisense)
`
`Target gene/cells
`
`siRNA
`concentr./
`time
`
`siRNA activity (% of
`degraded mRNA or
`silenced protein)
`
`Ref.
`
`hCav
`
`PS4
`
`hCav
`
`PS8
`
`hCav
`
`PS12
`
`hCav
`
`PS16
`
`hCav
`
`PS21
`
`Thio siRNA-2a
`
`Thio siRNA-2b
`
`Thio siRNA-2c
`
` oooooooooooooooooooTT
`
`TTooooooooooooooooooo
`
` oooooooooooooooooooTT
`
`TTooooooooooooooooooo
`
`oooooooooooooooooooTT
`
`TTooooooooooooooooooo
`
`oooooooooooooooooooTT
`
`TTooooooooooooooooooo
`
`oooooooooooooooooooTT
`
`TTooooooooooooooooooo
`
`oooooooooooooooooooUU
`
`UUooooooooooooooooooo
`
`oooooooooooooooooooUU
`
`UUooooooooooooooooooo
`
`oooooooooooooooooooUU
`
`UUooooooooooooooooooo
`
`>95 (>95% native siRNA)
`
`~ 93
`
`~90
`
`~85
`
`~75
`
`95 (82% native siRNA)
`
`86
`
`87
`
`[70]
`
`[72]
`
`hCav/HeLa
`
`200 nM/
`
`48 h
`
`Luc/HeLa
`
`10 nM/
`48 h
`
`Thio siRNA-2d
`
`Thio siRNA-2e
`
`Thio siRNA-2f
`
`SS/AS-P-S
`
`SS-P-S/AS
`
`DS-P-S
`
`hTF167i
`
`P1+1
`
`hTF167i
`
`P0+2
`
`hTF167i
`
`P2+2
`
`hTF167i
`
`P2+4
`
`oooooooooooooooooooUU
`
`UUooooooooooooooooooo
`
`oooooooooooooooooooUU
`
`UUooooooooooooooooooo
`
`oooooooooooooooooooUU
`
`UUooooooooooooooooooo
`
`oooooooooooooooooooTT
`
`TTooooooooooooooooooo
`
`oooooooooooooooooooTT
`
`TTooooooooooooooooooo
`
`oooooooooooooooooooTT
`
`TTooooooooooooooooooo
`
`oooooooooooooooooooUA
`
`GCooooooooooooooooooo
`
` oooooooooooooooooooUA
`
`GCooooooooooooooooooo
`
` oooooooooooooooooooUA
`
`GCooooooooooooooooooo
`
` oooooooooooooooooooUA
`
`GCooooooooooooooooooo
`
`94
`
`84
`
`81
`
`42 (95% native siRNA)
`
`62
`
`47
`
` ~ 88 (90% native siRNA)
`
`~ 88
`
`~ 82
`
`~ 80
`
`[71]
`
`[41]
`
`EGFP-C1/
`
`HeLa
`
`50 nM/
`
`42 h
`
`TF/HaCaT
`
`100 nM/
`
`24 h
`
`

`

`920 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 9
`
`Nawrot and Sipa
`
`Site of modification (underlined)
` 5’fi
` 3’ (sense)
` 3’‹
` 5’ (antisense)
`
`Target gene/cells
`
`siRNA
`concentr./
`time
`
`activity (%) of degraded
`mRNA or silenced
`protein)
`
`Ref.
`
`(Table 1) Contd….
`
`Duplex ID
`
`s/as-ps(even#)
`
`s/as-ps(19-21)
`
`s/as-ps(2)
`
`s-ps(even#)
`
`/as
`
`s-ps(19-21) /as
`
`s-ps(2) /as
`
`s-ps(even#)
`
`/as-ps(even#)
`
` oooooooooooooooooooTT
`
`TTooooooooooooooooooo
`
` oooooooooooooooooooTT
`
`TTooooooooooooooooooo
`
` oooooooooooooooooooTT
`
`TTooooooooooooooooooo
`
` oooooooooooooooooooTT
`TTooooooooooooooooooo
`
` oooooooooooooooooooTT
`
`TTooooooooooooooooooo
`
` oooooooooooooooooooTT
`
`TTooooooooooooooooooo
`
` oooooooooooooooooooTT
`
`TTooooooooooooooooooo
`
`Lamin A/C / 3T3
`
`100 nM /
`
`68 h
`
`~90 (90% native siRNA)
`
`~ 87
`
`~85
`
`~90
`
`~90
`
`~90
`
`~87
`
`~80
`
`~ 85
`
`[44]
`
`s-ps(19-21)/as-ps(19-21)
`
` oooooooooooooooooooTT
`
`s-ps(2)/as-ps(2)
`
`TTooooooooooooooooooo
`
` oooooooooooooooooooTT
`
`TTooooooooooooooooooo
`
`s-ps(19-21)/as-ps(19-21)
`
` oooooooooooooooooooTT
`
`TTooooooooooooooooooo
`
`Lamin A/C /
`HeLa
`
`100 nM /
`
`~95 (~95% native siRNA)
`
`46 h
`
`charge) [76] and were two times more resistant towards
`nucleases than their PS-counterparts. Their activity in green
`fluorescent protein (GFP) silencing was found equal or
`higher as compared to the native siRNAs, and higher in
`respect
`to
`the corresponding PS-siRNAs, unless
`the
`modifications were present in the middle of the antisense
`strand. Notably, the boranophosphate analogues were non-
`toxic for cells, even when 76 % of the internucleotide bonds
`underwent modification [75].
`
`C2’-Ribose Modifications [Fig. (4)]
`
`Increased stability against nucleases can result from
`modification or substitution the 2’-OH group in the ribose
`ring of the ribonucleotide units of siRNA. As demonstrated
`earlier in antisense strategy [77], alkylation of the 2’-OH
`group significantly enhances the stability of siRNA molecule
`and
`increases affinity of
`the modified strand
`into
`complementary unmodified RNA. Typically, the 2’-OH
`group is converted into 2’-O-methyl (2’-OMe) [31, 74], 2’-
`O-(2-(methoxy)-ethyl) (2’-OMOE) [41] groups or via 2’-
`acetoxy ethyl orthoester chemistry (ACE) into orthoester
`group [2’-O-CH(OCH2CH2OH)2] [74].
`Appreciated modification of the 2’-position of the ribose
`moiety involves 2’-OH to 2’-H substitution by application of
`2’-deoxyribonucleotides. In general, DNA oligomers are
`more resistant towards serum and intracellular nucleases,
`
`compared to oligoribonucleotides. Incorporation of DNA
`monomers at the ends of RNA molecules to the certain
`extent increases stability of the siRNA [29, 78].
`However, there are contradictory opinions about activity
`of so called hybrid siRNAs - constructs consisting of RNA
`strand hybridized with entire DNA strand. While some
`groups reported such hybrids as completely inactive [29, 78],
`others estimated them as even more potent than “pure RNA”
`duplexes [79, 80].
`In the case of highly stable siRNA duplexes containing
`fully modified 2’-OMe RNA strands (antisense as well as
`sense strand) there are many observations of a lost of their
`silencing function [31, 70]. The stability of siRNA constructs
`with 2’-OMe units in juxtaposed positions is comparable
`with those, containing fully modified strands, with retained
`ability to induce the RNAi process [31]. Similar effect was
`observed when several 2’-OMe-ribonucleotides were present
`at the 3’-ends of the RNA strands [41, 79-81]. The 2’-
`OMOE substituents are tolerated only if present in the sense
`strand. If they are located at the ends of the antisense strand,
`the siRNA activity is dramatically reduced, while their
`presence in the middle of the duplex does not affect
`significantly duplex activity [81]. The limited applicability
`of the 2’-O-alkyl substituents may be related to their steric
`hindrance causing distortion of the siRNA molecule from the
`preferred RNA A-type helical structure [31, 41].
`
`

`

`Chemical and Structural Diversity of siRNA Molecules
`
`Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 9 921
`
`The electronegative groups at the 2’-position strongly
`stabilize the C3’-endo conformation of the ribose ring,
`preferred for RNA strands [82 - 84], and modified in this
`way oligomers form exceptionally thermodynamically stable
`duplexes [70, 84]. RNA analogues containing 2’-fluoro-2’-
`deoxyribonucleotides are among the most prominent in
`respect to their thermodynamic stability and resistance
`against nucleases [68]. Promising pharmacokinetic and
`silencing results were obtained with constructs containing
`fluorine atom in the 2’-position, both in phosphate and
`phosphorothioate siRNA strands [44, 70, 71, 74, 81].
`Duplexes containing in 21-nt antisense strand all C and U
`residues (15 in total) replaced with 2’-fluoro-2’-deoxyuridine
`and 2’-fluoro-2’-deoxycytidine, and additionally possessing
`four such replacements in the sense strand, tested in HeLa
`cells were practically indistinguishable in their silencing
`activity from non-modified duplexes, even if they were
`modified in combination with phosphorothioate bonds [44].
`in vitro
`The 2’-F siRNA constructs were
`tested
`in
`lymphocytes-T culture without the use of transfecting agent.
`They were found to be stable towards RNase A and reduced
`HIV-1 viral infection [85]. The 2’-F siRNA worked well in
`vivo in mice co-transfected with GFP coding plasmids.
`Interestingly, despite of enhanced stability against nucleases,
`in this case no prolongation of gene silencing was observed
`[84].
`Distinct improvement of pharmacokinetics was observed
`for siRNAs carrying several different C2’ modifications
`introduced simultaneously. For example, high activity
`(>500-fold enhanced in comparison to the native siRNA) and
`improved stability was found for siRNA with all 2’-OH
`groups replaced in alternate positions with 2’-OMe and 2’-F
`substituents [32]. Constructs with additional modifications
`resulting from the presence of inverted abasic nucleosides at
`the ends of the sense strand effectively prevented HBV
`infection in mice [87, 88].
`Attractive group of modified siRNA molecules consists
`of 2’-O-arylated compounds with 2,4-dinitrophenyl group
`(DNP-RNA) [60]. The arylation could be done after
`enzymatic synthesis; the products were stable towards
`nucleases and did not require any carrier to cross the cellular
`membranes. Notably, they exert good RNAi activity and
`their cytotoxicity is rather low, making them good candidates
`for further evaluation [60].
`Some other modifications of 2’-position of ribose ring in
`siRNA units were tested including 2’-amino group [74, 78]
`and 2’-O-allyl group [41]. While introduction of 2’-amino-
`2’-deoxyuridine or cytidine units into antisense strand of
`siRNA triggered silencing activity, those modifications in
`the sense strand were well tolerated in RNA-induced gene
`silencing in C. elegans [74].
`An approach based on conformational restraints imposed
`on the ribose ring employing locked nucleic acids (LNA)
`[89] and ethylene bridged nucleic acids (ENA) [90] [Fig. (4)]
`has to be also mentioned. These bicyclic molecules differ in
`the number of carbon atoms in the bridges. LNA nucleotides
`possess one-, while ENA – two-carbon bridge. Initial studies
`showed
`that such modifications significantly enhance
`thermodynamic stability of the duplex (increase of the Tm
`parameter was 2-4 °C per modification). Surprisingly, the
`
`ENA units present at both ends of siRNA completely
`abolished its activity [42]. On the other hand, the presence of
`the LNA units in these positions seemed to be well accepted
`[70].
`Interestingly, observed activity of LNA-siRNA
`duplexes (which melt at the temperature > 95 °C), suggests
`high efficiency of the RISC helicase, which is able to unwind
`the strands of such unusually stable siRNA duplexes.
`
`Base Modifications Affecting siRNA Structure and
`Activity
`
`As demonstrated by Chiu and Rana in experiments
`performed with bulge siRNA strands in HeLa cells [43], the
`perfect A-type helix of siRNA is not required for effective
`interference. However, perfect mRNA/guide strand RNA
`helix recognition seems to be crucial for its assembly at
`RISC complex. Therefore, chemical modifications located in
`the guide (antisense) strand of siRNA, due to possible
`changes of the mRNA/guide siRNA local conformation, may
`tune gene silencing by RNA interference. Some nucleobases
`are known to affect the ribose conformation in similar way
`as substituents at 2’ position of the rib