J. Med. Chem. 2005, 48, 901-904
`
`901
`
`Fully 2¢-Modified Oligonucleotide
`Duplexes with Improved in Vitro
`Potency and Stability Compared to
`Unmodified Small Interfering RNA
`
`Charles R. Allerson,*,† Namir Sioufi,‡ Russell Jarres,‡
`Thazha P. Prakash,† Nishant Naik,† Andres Berdeja,§
`Lisa Wanders,§ Richard H. Griffey,†
`Eric E. Swayze,† and Balkrishen Bhat†
`Departments of Medicinal Chemistry, Antisense Lead
`Identification, and Research Chemistry,
`Isis Pharmaceuticals, 2292 Faraday Avenue,
`Carlsbad, California 92008
`Received October 18, 2004
`
`Abstract: We have identified a small
`interfering RNA
`(siRNA) motif, consisting entirely of 2¢ -O-methyl and 2¢ -fluoro
`nucleotides, that displays enhanced plasma stability and
`increased in vitro potency. At one site, this motif showed
`remarkable >500-fold improvement in potency over the un-
`modified siRNA. This marks the first report of such a potent
`fully modified motif, which may represent a useful design for
`therapeutic oligonucleotides.
`The specific and reversible modulation of gene ex-
`pression through the use of short synthetic oligonucle-
`otides has proven useful in the study of gene function
`and as a therapeutic mechanism in man.1 Recently,
`RNA interference (RNAi) has emerged as a novel
`mechanism that is activated in mammalian cells by
`small interfering RNAs (siRNAs), short RNA duplexes
`with strands of 21-23 nucleotides.2,3 Once inside the
`cell, siRNAs associate with proteins to form an RNA-
`induced silencing complex (RISC).4 A helicase activity
`associated with RISC separates the two strands of the
`duplex,5 releasing the sense strand and permitting the
`binding of the antisense strand to its messenger RNA
`(mRNA) target. The resulting duplex is a substrate for
`a RISC-associated nuclease, recently identified as Ago2
`(also known as eIF2C2 in man),6 which cleaves the
`target transcript at a single site.5,7
`Despite attempts to use siRNA in vivo,8-14 the reduc-
`tion of endogenous target mRNAs through the systemic
`delivery of siRNA has proven difficult. Although there
`have been conflicting reports on the nuclease stability
`of unmodified siRNA duplexes, there is evidence that
`many are degraded within minutes in mammalian
`serum.9,15,16 It seems likely that siRNAs with increased
`nuclease stability will have a better chance at eliciting
`an in vivo response. Efforts to determine the optimal
`use of stabilizing chemistries have been the focus of
`several recent reports.15,17-21 From these studies, the
`2¢ -O-methyl (2¢ -OMe) and 2¢ -deoxy-2¢ -fluoro (2¢ -F) modi-
`fications have shown promise in stabilizing siRNA
`without disrupting the efficiency of mRNA target reduc-
`tion. To further optimize the use of these and other
`chemistries in siRNA, we are conducting an extensive
`
`* To whom correspondence should be addressed. Phone: 760-603-
`4697. Fax: 760-603-4654. E-mail: callerson@isisph.com.
`† Department of Medicinal Chemistry.
`‡ Department of Antisense Lead Identification.
`§ Department of Research Chemistry.
`
`SAR analysis of chemically modified siRNA. One of the
`most promising designs from these studies is a fully
`modified duplex that consists of alternating 2¢ -OMe and
`2¢-F nucleotides. Surprisingly, duplexes with this sub-
`stitution display increased in vitro potency and in-
`creased stability. Here, we report on this remarkably
`active motif and present our preliminary findings.
`The duplexes used in our SAR study were designed
`to target one of two sites within the coding region of
`the human PTEN mRNA, both previously reported as
`valid target sites for siRNA.22 Modified and unmodified
`duplexes were introduced into HeLa cells using a
`cationic lipid transfection reagent (lipofectin). In vitro
`activity was measured by performing RT-PCR on the
`PTEN mRNA and comparing to mRNA levels of un-
`treated cells. All PTEN signals were normalized to total
`RNA, as measured with RiboGreen.23
`Natural Dicer-generated siRNAs have 2-nucleotide 3¢
`overhangs on both ends of the short duplex.2 In an effort
`to mimick this design, most synthetic siRNAs are
`designed with 3¢ overhangs, typically with the sequence
`dTdT. Several recent reports have provided evidence
`that these overhangs bind to the PAZ domains found
`in numerous proteins, including the Ago2 component
`of RISC, perhaps functioning as a specificity determi-
`nant.24-28 However, there have been other reports that
`blunt-ended RNA duplexes can function equally well
`and may be more stable to exonucleolytic degradation.21
`Given our goal of developing duplexes with optimal
`stability and activity, we explored the use of blunt-ended
`duplexes at both PTEN target sites (sites A and B). The
`activities of 19-base-pair duplexes having 3¢ -dTdT over-
`hangs (1 and 4) were compared to those of otherwise
`identical blunt-ended duplexes (2 and 5) in a 10-point
`dose-response experiment (Figure 1). We found only a
`minimal impact on activity with the removal of the
`overhangs. To confirm the specificity of PTEN mRNA
`reduction, duplexes containing six mismatches (3 and
`6) to the target site were included as negative controls
`in each experiment. Neither control affected PTEN
`mRNA or total RNA levels.
`After validation of the use of blunt-ended constructs,
`the remaining SAR was performed without the use of
`3¢ overhangs. Among the duplex designs examined were
`several that belonged to a class of “alternating” motifs
`in which one type of modified nucleotide was placed in
`alternating positions with a ribonucleotide or a different
`modified nucleotide. In a recent publication, motifs of
`this type, using 2¢-OMe and unmodified (2¢-OH) nucle-
`otides, were shown to have enhanced serum stability
`and single dose in vitro activities similar to those of the
`corresponding unmodified blunt-ended duplexes.21 Dur-
`ing the course of our studies, we examined siRNAs with
`the same 2¢-OMe/2¢-OH substitution. We found that
`duplexes with this motif had in vitro potencies similar
`to those of unmodified siRNAs (see Supporting Informa-
`tion). As part of our broader SAR studies, however, we
`also examined the effect of combining the 2¢-OMe
`substitution with other chemistries, such as 2¢ -F, which
`has already been shown to be well-tolerated in siRNA.15
`This led to the identification of our most potent con-
`
`10.1021/jm049167j CCC: $30.25 © 2005 American Chemical Society
`Published on Web 01/20/2005
`
`Alnylam Exh. 1016
`
`

`

`902 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
`
`Letters
`
`Site A
`AAGUAAGGACCAGAGACAAdTdT-3'
`5•.
`3'-dTdTUUCAUUCCUGGUCUCUGUU
`-5'
`AAGUAAGGACCAGAGACAA
`5•.
`UUCAUUCCUGGUCUCUGUU
`3'·
`s·-
`3'-
`
`-3'
`-5'
`
`-3'
`-5'
`
`AAGiAAiGAWuG~GA~AA
`UUC UU CU UC CU UU
`
`2
`
`3
`
`A
`
`140
`
`120
`
`! 100
`e
`~ 80
`
`80 J 40
`
`20
`
`0 ...J....L...1.-'-~-'-'-'--'-'--'-....... -'-.L...l.-'-~-'-'-'--'-'--'-....... -'-.L...1....J...L..l.-'-L...J....L.J..J
`~~~~,§§~~~:~:~i! §§~~~~~:~~! §§S~~~q~~~~
`0000
`0000
`OOOOOON~
`-
`3
`
`2
`
`SiteB
`
`4
`
`5
`
`6
`
`s·-
`GGGUAAAUACAUUCUUCAUdTdT-3'
`3'-dTdTCCCAUUUAUGUAAGAAGUA
`-5'
`s·-
`GGGUAAAUACAUUCUUCAU
`3'-
`CCCAUUUAUGUAAGAAGUA
`GGGAAA~UAW.UU~UUUAU
`S'-
`3'-
`CCCUUU£AUA!,.AA£AAAUA
`
`-3'
`-s·
`-3'
`-5'
`
`B
`
`140
`
`120
`
`" ~ 100
`e
`z 80
`t:
`c 60
`~ 40
`if
`
`20
`
`4
`5
`6
`Figure 1. Reduction of endogenous PTEN mRNA in HeLa
`cells by siRNAs having 3¢ -dTdT overhangs or blunt ends. HeLa
`cells were transfected with siRNAs at the indicated concentra-
`tions in the presence of lipofectin and treated for 20 h followed
`by lysis and RT-PCR. Message levels are reported as percent
`of PTEN mRNA from untreated cells. The bottom strand of
`each duplex is complementary to the target mRNA (mis-
`matches to the target site are indicated with an underline).
`(A) PTEN mRNA reduction by siRNAs targeted to site A. Cells
`were also treated with a duplex containing six mismatches to
`the target site as a negative control (3). (B) PTEN mRNA
`reduction by siRNAs targeted to site B. Cells were also treated
`with a duplex containing six mismatches to the target site as
`a negative control (6).
`
`struct design, in which both strands were substituted
`with alternating 2¢-OMe and 2 ¢-F nucleotides (Figure
`2). From an eight-point dose-response analysis in HeLa
`cells, we were able to estimate IC50 values of each duplex
`(Table 1; see also Supporting Information).
`At site A, the duplex with alternating 2¢-F/2¢-OMe
`chemistry (7, Figure 2) had biological activity that was
`roughly equivalent to that of the parent siRNA (2). At
`site B, however, the 2¢-F/2¢-OMe duplex ( 10) displayed
`dramatically improved potency. Even at the lowest
`concentration (2 pM), target reduction was greater than
`75% relative to untreated control, minimally reflecting
`500-fold improvement in potency over the unmodified
`siRNA (5). Throughout these studies, we were cognizant
`of the importance of the 5¢ -phosphates normally present
`on Dicer-generated siRNA duplexes. The 5¢-phosphate
`on the antisense strand has been shown to be critical
`for efficient assembly and activation of RISC in mam-
`malian systems.29 However, it has been shown that
`
`A
`
`120
`
`100
`
`80
`
`"' z
`0: e
`z
`t: 80
`! 40
`i 20
`
`B
`
`120
`
`2
`
`7
`
`8
`
`9
`
`-3'
`.5•
`
`SlteA
`5'- AAGUAAGGACCAGAGACAA
`3'- UUCAUUCCUGGUCUCUGUU
`S'- AAGUAAGGACCAGAGACAA -3'
`3'- UUCAUUCCUGGUCUCUGUU -s· N: 2'-0Me
`N=2'-F
`S'-P-AAGUAAGGACCAGAGACAA -3'
`P • 5'aphosphate
`3'- UUCAUUCCUGGUCUCUGUU.P-5'
`s·- AAGCAACGAGAAGCGAUAA
`-3'
`3'- UUC~ UU~CUJ.ll.UC~CUalJU -5'
`
`2
`
`7
`
`8
`
`9
`
`5
`
`10
`
`11
`
`12
`
`Site B
`-3'
`S'- GGGUAAAUACAUUCUUCAU
`3•. CCCAUUUAUGUAAGAAGUA
`-5'
`s·- GGGUAAAUACAUUCUUCAU -3'
`3•. CCCAUUUAUGUAAGAAGUA -5'
`S'-P-GGGUAAAUACAUUCUUCAU -3'
`3'- CCCAUUUAUGUAAGAAGUA.P-5'
`s·- ggg~~~~~~~~~l -3'
`
`3'-
`
`-5'
`
`N = 2'-0Mc
`N = 2'-F
`P - S'.phosphate
`
`100
`
`80
`
`80
`
`" z
`0: e
`z
`"'
`Ii: I 40
`
`0.
`
`20
`
`ONCO .... NOOOO
`
`!!!~ONIDIO~
`
`10
`11
`12
`5
`Figure 2. Reduction of endogenous PTEN mRNA in HeLa
`cells by unmodified or fully 2¢ -F/2¢ -OMe modified 19-base-pair
`oligonucleotide duplexes. HeLa cells were transfected with
`siRNAs at the indicated concentrations in the presence of
`lipofectin and treated for 20 h followed by lysis and RT-PCR.
`Message levels are reported as percent of PTEN mRNA from
`untreated cells. The bottom strand of each duplex is comple-
`mentary to the target mRNA (mismatches to the target site
`are indicated with an underline). The 2¢-F modification is
`indicated in green, the 2¢-OMe modification is indicated in
`purple, while the presence of a 5¢-phosphate is indicated by
`the letter “P”. (A) PTEN mRNA reduction by duplexes targeted
`to site A. (B) PTEN mRNA reduction by duplexes targeted to
`site B.
`
`Tm (°C)
`72.8
`93.9
`nd
`62.0
`nd
`82.0
`
`plasma half-life (min)
`(cid:24)30
`nd
`>420
`nd
`nd
`>420
`
`Table 1. Summary of in Vitro Activity, Plasma Stability, and
`Duplex Thermal Stabilitya
`duplex
`site
`IC50 (nM)
`2
`A
`0.26
`7
`A
`0.31
`8
`A
`0.068
`5
`B
`0.81
`<0.002
`10
`B
`<0.002
`11
`B
`a nd ) not determined.
`prephosphorylation is generally unnecessary because
`the siRNAs are phosphorylated by endogenous kinases.
`Anticipating that chemically modified duplexes might
`be less efficiently processed by these kinases,30 we
`compared the activities of 2¢ -F/2¢ -OMe duplexes with
`preestablished 5¢-phosphates (Figure 2, 8 and 11). At
`site A, addition of a synthetic 5¢ -phosphate produced
`
`

`

`Letters
`
`Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 903
`
`modest improvement (5-fold) in potency (Table 1). Given
`the already potent activity of 10, we were unable to
`resolve any beneficial effect of 5¢ -phosphorylation at site
`B. Similar effects from phosphorylation have been
`observed with this motif on other targets, where mod-
`estly active duplexes show improvement of in vitro
`potency upon addition of the 5¢ -phosphate, while the
`effect on extremely potent duplexes is difficult to resolve
`(data not shown). Furthermore, the 5¢ -phosphate on the
`antisense strand appears to be largely responsible for
`this improvement in potency, although the magnitude
`of the effect may depend on cell type (data not shown).
`We also prepared and tested modified duplexes that
`contained six mismatches to either of the target sites
`(9 and 12). Neither mismatch-containing duplex pro-
`duced a significant reduction in PTEN mRNA or total
`RNA.
`To examine the relative serum stabilities of these
`duplexes, we tested unmodified siRNA 2 and 2¢ -F/2¢ -
`OMe modified duplexes 8 and 11 for their ability to
`resist degradation in mouse plasma. Each of the du-
`plexes was treated with 25% mouse plasma at 37 °C
`for up to 7 h. At various time points, aliquots were
`removed and examined for intact duplex by capillary
`gel electrophoresis.31,32 From these measurements, we
`plotted percent intact duplex against time and assessed
`the relative stabilities of the three duplexes (Figure 3A,
`Table 1). From this straightforward analysis, the stabil-
`ity of the 2¢ -F/2¢ -OMe duplexes was striking. Even after
`7 h, the duplexes were greater than 60% (8) or 70% (11)
`intact. While there was an initial loss of duplex in each
`case, this may correspond to rapid loss of imperfectly
`annealed strands. Regardless, the remaining duplex
`degrades at a rate that suggests a half-life of much
`greater than 7 h.
`It has previously been shown that the use of 2¢-F
`modifications can increase the thermal stability of
`oligonucleotide duplexes.33 Because duplexes 7, 8, 10,
`and 11 each contain a total of 19 2¢-F nucleotides, we
`anticipated that these duplexes might have much
`greater thermal stabilities than their unmodified coun-
`terparts. To examine this possibility, we measured the
`thermal stabilities of two control duplexes (2 and 5) and
`two modified duplexes (7 and 11) (Figure 3B). In each
`case we see a roughly 20 °C increase in Tm with the
`fully modified duplexes. This corresponds to slightly
`more than 1 °C increase in Tm per 2¢ -F substitution. This
`increase in thermal stability is likely to explain in part
`the enhanced plasma stability of the modified duplexes
`and may improve the interaction between the antisense
`strand and the target mRNA.
`It is surprising that duplexes with such high thermal
`stabilities can function so efficiently and potently rela-
`tive to the unmodified siRNAs. Recent studies have
`suggested that RISC chooses which strand to retain on
`the basis of differences in thermodynamic stability at
`the 5¢ ends of the strands, with the strand having the
`5¢ end of lower stability being more likely to load into
`RISC.34,35 It is worth noting that one end of the site B
`duplex contains three consecutive G-C base pairs.
`Although the introduction of 2¢-F nucleotides should
`raise the thermodynamic stability of both ends of the
`duplex, it may increase the already more-stable end
`above a critical threshold that the RISC-associated
`
`A
`
`100
`
`~
`
`80
`
`60
`
`><
`GI
`Q.
`:I
`C
`tl
`J!
`.5
`c 40
`GI
`I:?
`GI
`D.
`
`20
`
`0
`
`0
`
`60
`
`120
`
`180
`
`240
`
`300
`
`360
`
`420
`
`Time (minutes)
`
`B 1.4
`
`(.)
`
`GI 1.3
`C .,,
`-e
`0
`ti) 1.2
`.a cc
`"C -~ ii 1.1
`E ...
`0 z
`
`1.0
`
`0.9
`
`•
`(cid:127)
`...
`,.
`
`2
`7
`5
`11
`
`20
`
`30
`
`40
`
`50
`
`60
`
`70
`
`80
`
`90
`
`100
`
`Temperature (degrees C)
`Figure 3. Comparison of the biophysical properties of un-
`modified blunt-ended siRNAs and 2¢ -F/2¢ -OMe modified
`duplexes. (A) Stability of duplexes in 25% (v/v) mouse plasma.
`Duplexes were treated with 25% mouse plasma at 37 °C for
`the indicated times, then examined by capillary gel electro-
`phoresis to determine the amount of intact duplex (see
`Supporting Information for details). The plots shown are the
`result of two independent experiments. (B) Thermal denatur-
`ation profiles of both unmodified and modified duplexes in 100
`mM NaCl, 10 mM sodium phosphate, pH 7.5, 0.1 mM EDTA,
`and 4 (cid:237)M of each strand. Absorbance at 260 nm was measured
`as the temperature was raised from 15 to 85 °C or from 30 to
`95 °C. Absorbances were normalized to facilitate graphical
`depiction. Melting temperatures (Tm) were calculated from
`first-derivative curves of at least two separate experiments and
`are summarized in Table 1.
`helicase cannot overcome, shifting the bias for strand-
`loading more in favor of the antisense strand. We also
`observed that among the unmodified duplexes, the best
`activity comes from the duplex having the highest Tm,
`whereas the opposite is true for the 2¢-F/2¢-OMe motif.
`Although not definitive, these relationships may hint
`at the existence of an optimal thermal stability.
`From these observations, the alternating 2¢ -F/2¢ -OMe
`motif appears to be an attractive design for creating
`functionally active and stable RNA duplexes. Clearly,
`these duplexes potently reduce levels of endogenous
`target mRNA, with the addition of a 5¢ -phosphate to the
`antisense strand further enhancing in vitro potency. The
`biophysical properties of this duplex motif, reflected in
`its enhanced serum and thermal stability, also favor its
`chances at surviving in serum, which will hopefully
`
`

`

`904 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
`
`translate to improved in vivo potency. The identification
`of this motif from our SAR analysis highlights the value
`of screening oligonucleotides containing multiple chem-
`istries and may prove a useful strategy in related areas
`such as micro-RNA. Although promising, the utility and
`optimal design of this motif for in vivo applications and
`a biochemical explanation for the remarkable increase
`in potency remain to be determined and are the focus
`of ongoing studies. We anticipate that this and other
`chemically modified duplex motifs will ultimately prove
`useful in the design of clinically active nucleic acids.
`
`Acknowledgment. The authors thank Bruce Ross,
`Mystie Nguyen, and Mingming Han for providing the
`2¢ -O-methyl and 2¢ -deoxy-2¢ -fluoro nucleoside phos-
`phoramidites, Hans Gaus, Sam Lee, and Jodee Stein-
`berg for their assistance in oligonucleotide analysis, and
`Prasad Dande, Sue Freier, Bridget Lollo, and Brenda
`Baker for their thoughtful discussions about this project.
`
`Supporting Information Available: Methods for the
`synthesis and purification of the oligonucleotides, mass
`spectral and capillary gel electrophoresis data of each com-
`pound, and details of the biological and biochemical assays.
`This material is available free of charge via the Internet at
`http://pubs.acs.org.
`
`References
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