Biophysical Journal Volume 100 June 2011 2981–2990
`
`2981
`
`Fluorescence Cross-Correlation Spectroscopy Reveals Mechanistic
`0
`Insights into the Effect of 2
`-O-Methyl Modified siRNAs in Living Cells
`
`‡6
`
`†
`
`†
`
`†6
`Jo¨ rg Mu¨ tze,
`and Petra Schwille
`Markus Landthaler,
`Karin Crell,
`Wolfgang Staroske,
`Thomas Ohrt,
`‡
`†
`Department of Cellular
`Department of Biophysics, Biotechnology Center, Dresden University of Technology, Dresden, Germany;
`§
`Berlin Institute for Medical Systems Biology, Berlin,
`Biochemistry, Max Planck Institute of Biophysical Chemistry, Goettingen, Germany; and
`Germany
`
`§
`
`†
`
`*
`
`ABSTRACT RNA interference (RNAi) offers a powerful tool to specifically direct the degradation of complementary RNAs, and
`thus has great therapeutic potential for targeting diseases. Despite the reported preferences of RNAi, there is still a need for new
`techniques that will allow for a detailed mechanistic characterization of RNA-induced silencing complex (RISC) assembly and
`activity to further improve the biocompatibility of modified siRNAs. In contrast to previous reports, we investigated the effects of
`0
`0
`2
`-O-methyl (2
`OMe) modifications introduced at specific positions within the siRNA at the early and late stages of RISC
`0
`assembly, as well as their influence on target recognition and cleavage directly in living cells. We found that six to 10 2
`OMe
`0
`nucleotides on the 3
`-end inhibit passenger-strand release as well as target-RNA cleavage without changing the affinity, strand
`0
`0
`OMe modifications introduced at the 5
`-end reduced activated RISC stability, whereas incor-
`asymmetry, or target recognition. 2
`porations at the cleavage site showed only minor effects on passenger-strand release when present on the passenger strand.
`Our new fluorescence cross-correlation spectroscopy assays resolve different steps and stages of RISC assembly and target
`recognition with heretofore unresolved detail in living cells, which is needed to develop therapeutic siRNAs with optimized in vivo
`properties.
`
`INTRODUCTION
`
`RNA interference (RNAi) is an evolutionary conserved gene-
`silencing mechanism that uses short, double-stranded RNAs
`(dsRNAs) to identify complementary target RNAs for
`sequence-specific degradation (1). The RNAi mechanism
`requires a ribonucleoprotein complex known as the RNA-
`induced silencing complex (RISC), which is composed of
`an Argonaute (Ago) protein associated with a short, single-
`stranded RNA of 19–30 nucleotides (nt). These short guide
`RNAs originate from endogenous or exogenous dsRNA
`substrates of various secondary structures (2–5). To function
`as guiding cofactors, the short dsRNAs are bound by the
`RISC loading complex (RLC), which consists of Dicer,
`TRBP, and a member of the Argonaute protein family
`(4,6,7). The selection of the latter guide RNA is governed
`by the thermodynamic stability of the duplex termini (8).
`For target recognition, RISC is guided by single-stranded
`RNA on the basis of sequence complementarity. In the
`current two-state model, not all positions of the guide RNA
`participate equally in target recognition (9,10). Therefore,
`nucleotides 2–8 of the guide strand (termed the seed
`sequence) mediate the primary interaction with the target
`RNA. Once the seed sequence primes to the target, the duplex
`0
`-end, leading to a con-
`formation propagates toward the 3
`formational change in Argonaute (11–13). Depending on
`the extent of complementarity in conjunction with the asso-
`ciated Argonaute family member, RISC can trigger gene
`
`Submitted March 9, 2011, and accepted for publication May 5, 2011.
`6
`
`Thomas Ohrt and Wolfgang Staroske contributed equally to this work.
`
`*Correspondence: schwille@biotec.tu-dresden.de
`
`Editor: Paul W. Wiseman.
`Ó 2011 by the Biophysical Society
`0006-3495/11/06/2981/10 $2.00
`
`translational
`cleavage,
`expression by endonucleolytic
`repression, deadenylation, decapping, and/or translocation
`into P-bodies (reviewed in Chekulaeva and Filipowicz (14)).
`One class of small, silencing RNAs is composed of short
`interfering RNAs (siRNAs), which consist of a dsRNA stem
`0
`-phosphate and 2 nt overhangs at
`of ~19 nt containing a 5
`0
`-ends (3,5). The identification of siRNAs resulted
`both 3
`in the development of chemically synthesized siRNAs to
`specifically trigger the degradation of targeted mRNA tran-
`scripts in mammalian cells (15). Recent advances in chem-
`istry and oligonucleotide synthesis lead to the development
`of therapeutic siRNAs with improved pharmacokinetic and
`pharmacodynamic properties (16–19). Therefore, various
`modifications, such as the phosphorothioate (PTO) back-
`0
`0
`0
`-O-methyl (2
`-OMe) and 2
`-fluoro
`bone modification and 2
`0
`F) sugar modification, have been comprehensively eval-
`(2
`uated for their use in siRNA-based medical
`therapies
`(18–24). The PTO modification is compatible with siRNA
`function, leads to an increased half-life in serum, and facil-
`itates cellular uptake; however, it also lowers thermal duplex
`stability, can display cytotoxic effects, and promotes non-
`specific protein binding. This is illustrated by the strong
`affinity to polyanion-binding proteins, which results in
`accumulation in the nucleus and P-bodies and can poten-
`tially cause unwanted side effects, thereby limiting its use
`in siRNA-mediated silencing (20–23,25).
`0
`F modification on pyrimidines
`The incorporation of the 2
`also stabilizes the siRNA duplex against nucleases without
`affecting the silencing activity, but also results in a strong
`localization in the nucleus (22–24).
`
`doi: 10.1016/j.bpj.2011.05.005
`
`Alnylam Exh. 1081
`
`

`

`2982
`
`Ohrt et al.
`
`0
`
`-OMe RNA modification is a naturally
`The nontoxic 2
`occurring RNA alteration that is found in mammalian tRNAs
`and rRNAs and does not alter siRNA subcellular localization
`0
`-OMe modi-
`(23). Furthermore, the substitution of a single 2
`fication at position 2 of the guide strand reduces silencing of
`most off-target transcripts with complementarity to the seed
`region without affecting the silencing efficacy of the target
`0
`-OMe modification rates stabilize
`mRNA (26). Increased 2
`the duplex against nucleases, but can reduce or completely
`0
`-OMe
`abolish silencing activity of siRNAs (22,23,27). 2
`modifications on nucleotides 18–21 resulted in increased
`0
`-OMe modifi-
`silencing rates after 48 h (23). Although the 2
`cation can result in inactive duplexes or silencing of inhibited
`duplexes, it provides a natural and powerful tool that can be
`used to specifically increase the lifetime, specificity, and
`potency of siRNAs needed for RNAi-based pharmaceuticals
`(20,22,27). So far, investigators have studied the effects of
`0
`-OMe modification on siRNA-mediated silencing by clas-
`2
`sical silencing readouts, either by targeting an endogenous
`gene analyzed by Western blot or quantitative reverse tran-
`scription-polymerase chain reaction, or by standard reporter
`assays such as mRFP/EGFP expression or the dual luciferase
`assay (20–24,27). Still, little is known about the mechanism
`0
`-OMe nucleotides, which can
`of silencing inhibition by 2
`affect factors such as the affinity to RISC, strand separation,
`cleavage activity, and the stability of the incorporated guide
`strand. In addition, the ability to directly analyze the RNAi
`mechanism in living cells in real time at the early phase of
`RISC activation (1–6 h) (28) could result in a more detailed
`characterization of modified siRNAs, which is needed to
`further improve RNAi-based pharmaceuticals.
`We recently established fluorescence cross-correlation
`spectroscopy (FCCS) assays that allowed us to investigate
`the origination of nuclear RISC in a living cell in real time
`with single-molecule sensitivity (28). The FCCS method
`has a high sensitivity and makes it possible to directly access
`strand separation, guide-strand incorporation (including the
`duration of incorporation), and RISC-target-RNA interaction
`independently of reporter systems, translational regulation,
`and knock-down of an mRNA or protein, which require
`longer incubation times and cannot locate the individual
`step of silencing inhibition.
`-OMe modi-
`Here, we report on the positional effects of 2
`0
`0
`-end, 3
`-end, and at the cleavage
`fications in siRNAs on the 5
`site of either the guide or passenger strand or in combination.
`By applying FCCS, we were able to perform this character-
`ization in real time and in different cellular compartments.
`We show for the first time, to our knowledge, that stretches
`of modifications longer than 4 nt inhibit strand separation
`and increase the lifetime of the RISC-target interaction by
`inhibiting the cleavage activity of Ago2, and that modifica-
`0
`-end results in the destabilization of the
`tion of the 5
`0
`Ago2–guide-strand interaction. Internal 2
`-OMe modifica-
`tions display only marginally inhibitory effects on strand
`separation and cleavage activity.
`
`0
`
`Biophysical Journal 100(12) 2981–2990
`
`MATERIALS AND METHODS
`
`Cell culture
`
`ER293 cells stably transfected with the pERV3 vector (Stratagene, Santa
`
`C in DMEM (high glucose; Sigma. St. Louis,
`Clara, CA) were cultured at 37
`MO) with 10% fetal calf serum (FCS; PAA Laboratories GmbH, Pasching,
`Austria), 2 mM glutamine (Gibco, Carlsbad, Ca), and 0.3 mg/ml G418
`(50 mg/ml; Gibco).
`The cell line 10G, which stably expresses EGFP-Ago2 (introduced by
`
`C in Dulbecco’s modified Eagle’s
`Ohrt et al. (28)), was cultured at 37
`medium (high glucose; Sigma) with 10% FCS, 2 mM glutamine, 0.3 mg/ml
`G418, and 0.4 mg/ml Hygromycin B. All cells were regularly passaged at
`subconfluency and plated at a density of 1–5  104 cells/ml.
`
`Microinjection
`For microinjection, 5–10  104 10G cells were transferred onto MatTek
`chambers coated with Fibronectin (25 mg/ml in phosphate-buffered saline
`including CaCl2 and MgCl2; Roche, Basel, Switzerland) 24 h before micro-
`injection. The micropipette (Femtotip 2; Eppendorf, Hamburg, Germany)
`was loaded with 1.5–4 mM of labeled siRNAs in 110 mM K-gluconate,
`18 mM NaCl, 10 mM HEPES pH 7.4, and 0.6 mM MgSO4. The microma-
`nipulator consisted of a FemtoJet and InjectMan NI2 mounted directly on
`a microscope. The working pressure for injection was 20–40 hPa for
`0.1 s and a holding pressure of 15 hPa.
`
`FCCS setup
`
`FCCS and laser scanning microscopy were carried out on a commercial system
`consisting of an LSM510 and a ConfoCor3 (Zeiss, Oberkochen, Germany).
`The 488 nm line of an Ar-Ion laser and the 633 nm line of a HeNe laser
`were attenuated by an acousto-optical tunable filter to 3.5 kW/cm2 and
`1.05 kW/cm2, and directed via a 488/633 dichroic mirror onto the back aper-
`ture of a Zeiss C-Apochromat 40, N.A.¼ 1.2, water immersion objective.
`Fluorescence emission light was collected by the same objective and split
`into two spectral channels by a second dichroic (LP635). To remove any
`residual laser light, a 505–610 nm bandpass or 655 nm longpass emission filter,
`respectively, was employed. The fluorescence was recorded by avalanche
`photodiode detectors (APDs) in each channel. For EGFP-Ago2 autocorrelation
`measurements, a mirror was substituted for the second dichroic and a 505 nm
`longpass emission filter was used in a one-channel setup. Out-of-plane fluores-
`cence was reduced with a 70 mm pinhole. The fluorescence signals were
`software-correlated and evaluated with MATLAB (The MathWorks, Natick,
`MA) via a weighted Marquardt nonlinear least-square fitting routine.
`Laser scanning microscopy was performed with the APDs of the Con-
`foCor3 on the same setup. Cell measurements were performed in air-buffer
`(150 mM NaCl, 20 mM HEPES pH 7.4, 15 mM glucose, 150 mg/ml bovine
`serum albumin, 20 mM Trehalose, 5.4 mM KCl, 0.85 mM MgSO4, 0.6 mM
`CaCl2) at room temperature.
`
`RESULTS
`
`0
`
`Establishment of delivery and FCCS compatibility
`conditions of modified siRNAs
`0
`
`F RNA modifications
`-OMe, PTO, and 2
`We investigated 2
`(the most commonly used and characterized RNA modifica-
`tions) for their compatibility with our recently published
`intracellular FCCS assay (28). Differently modified Cy5-
`labeled siRNAs were microinjected into ER293 cells, and
`their subcellular localization and diffusion was studied in
`0
`F
`living cells. As described previously (23), the PTO and 2
`
`

`

`Effects of 2
`
`0
`
`-OMe Modified siRNAs
`
`2983
`
`and measured the cross-correlation amplitudes after dif-
`ferent incubation times. The incorporation of the labeled
`strand resulted in increased cross-correlation amplitudes
`(Fig. 1 A, red curve), whereas the incorporation of the non-
`labeled strand led to low cross-correlation amplitudes
`(Fig. 1 A, black curve).
`To investigate the RISC-target interaction, the EGFP-
`Ago2 expressing cell line was transfected with nonlabeled
`siRNAs and microinjected with a 50-nt-long, Cy5-labeled
`target RNA 24 h after transfection (Fig. 1 B). The stable inter-
`action of RISC with the target RNA resulted in increased
`cross-correlation amplitudes (Fig. 1 B, red curve), whereas
`RISC-mediated cleavage or no interaction led to low cross-
`correlation amplitudes (Fig. 1 B, black curve) (28).
`
`0
`
`0
`-end
`-OMe modifications on the 3
`Increased 2
`inhibit strand separation and target RNA cleavage
`0
`
`-OMe-
`To study the incorporation and strand separation of 2
`modified siRNAs intracellularly, we used siTK3 siRNA,
`which targets the mRNA of Renilla luciferase encoded on
`the plasmid pRL-TK but does not show any sequence
`homology with endogenous mRNA transcripts. We calcu-
`0
`-end hybridization energy of siTK3 as described
`lated the 5
`previously (8) to define the guide and passenger strand of
`the siTK3 duplex (Fig. 2 A). We used siTK3 siRNA because
`it displays a strictly asymmetric incorporation of the guide
`
`Incorporation of
`labelled strand
`
`A
`Ago2
`
`+
`
`EGFP
`
`Ago2
`
`EGFP
`
`B
`
`Ago2
`
`EGFP
`
`35
`
`30
`
`25
`
`20
`
`15
`
`10
`
`05
`
`EGFP-Ago2 bound to target-RNA in %
`
`+
`
`Interaction with
`target
`
`EGFP
`
`Ago2
`
`Incorporation of
`non-
`labelled strand
`
`1E-5 1E-4 1E-3 0,01
`τ/ ms
`
`0,1
`
`1
`
`10
`
`35
`
`30
`
`25
`
`20
`
`15
`
`10
`
`05
`
`EGFP-Ago2 interaction with labelled
`
`strand in %
`
`Ago2
`
`EGFP
`
`+
`
`Ago2
`
`EGFP
`
`Ago2
`
`EGFP
`
`No interaction
`with target
`no sequence
`complementarity
`
`+
`
`target-RNA
`cleavage
`
`Ago2
`
`EGFP
`
`1E-5 1E-4 1E-3 0,01
`τ/ ms
`
`0,1
`
`1
`
`10
`
`EGFP Ago2
`
`FIGURE 1 Illustration of the FCCS assays used in this study. (A) Incor-
`poration assay. An intracellular FCCS assay was used to study the incorpo-
`ration of the guide strand. The incorporation of the labeled guide strand
`results in increased cross-correlation amplitudes (red curve), whereas the
`incorporation of the nonlabeled passenger strand results in low cross-corre-
`lation amplitudes (black curve). (B) Target interaction assay. An intracel-
`lular FCCS assay was used to study the interaction of EGFP-RISC with
`its fluorescently labeled target RNA. A stable interaction can be observed
`for miRNAs that do not display RNA-cleavage activity, resulting in
`increased cross-correlation values (red curve), whereas no stable interaction
`can be observed for RISC complexes loaded with a guide RNA showing no
`sequence similarities or a guide strand leading to RISC-mediated target
`RNA cleavage (black curve).
`
`Biophysical Journal 100(12) 2981–2990
`
`modifications alter the subcellular localization of siRNAs
`from a predominantly cytoplasmic localization to a preferen-
`tially nuclear localization, as illustrated by a strong and
`punctuate accumulation of these siRNAs in the nucleus
`(see Fig. S1 A, lower panel in the Supporting Material). In
`previous experiments, we showed that unmodified siRNAs
`were excluded from the nucleus in an Exportin-5 (Exp5)-
`dependent process (29). To rule out the possibility that the
`predominantly nuclear localization was not due to reduced
`binding affinities of the modified duplexes to Exp5, we
`developed a two-color fluorescence RNA electrophoretic
`mobility shift assay (REMSA). Here, the affinity of modified
`siRNA duplexes (Cy5-labeled; red) to Exp5 in the presence
`of increasing amounts of unmodified competitor siRNA
`(Alexa488-labeled; green) is determined. The advantage of
`the two-color REMSA is that the amount of shifted com-
`petitor siRNA intensity can be measured and quantified
`simultaneously with the fluorescence intensity of the modi-
`fied duplexes. We found that modified and unmodified
`siRNA duplexes decreased in shifted fluorescence intensity
`in almost identical competitor-dependent manner (Fig. S2).
`This result indicates that the alteration of the subcellular
`0
`F- and PTO-modified siRNAs
`localization in the case of 2
`was not caused by changed affinities to Exp5, and most
`probably resulted from the previously reported affinity of
`0
`F modifications to polyanion-binding proteins
`PTO and 2
`concentrated in this organelle (25).
`–4-nt-long PTO- and
`Intracellular FCCS analysis of 3
`2
`F-modified siRNAs displayed strong bleaching rates of
`up to 70% for the Cy5-labeled siRNAs, especially in the
`nucleus, which made it impossible to obtain quantitative
`results (Fig. S1 B, lower panel). This strong photobleaching
`may arise from the increased dwell times of the siRNAs
`in the excitation volume caused by the aforementioned
`0
`F modifications to polyan-
`increased affinity of PTO and 2
`ion-binding proteins, which greatly reduces their mobility
`0
`-OMe-modified siRNAs conserved
`(25). In contrast, the 2
`the preferentially cytoplasmic localization independently
`0
`-OMe modifications
`of the position and quantity of the 2
`(Fig. S1 A, upper panel and Fig. S3) with moderate bleach-
`ing rates of 10–20% after 4 min (Fig. S1 B, upper panel).
`0
`-OMe modification is suitable for a
`Therefore,
`the 2
`detailed FCCS analysis of modified siRNAs in living cells
`to investigate position- and quantity-dependent effects on
`siRNA affinity to RISC, strand separation, asymmetry,
`guide-strand incorporation, the stability of the guide RNA-
`RISC interaction, target RNA recognition, and cleavage.
`
`0
`
`0
`
`FCCS assays for investigating the RNAi pathway
`in living cells
`
`To study the incorporation of the guide strand, asymmetry,
`and the stability of the RISC-guide RNA complex, we mi-
`croinjected Cy5-labeled siRNAs into an EGFP-Ago2 stable
`expressing cell line with endogenous expression levels (28)
`
`

`

`2984
`
`Ohrt et al.
`
`ΔG=-6,6 kcal/mol (as/guide strand)
`5'-UGAAUGGUUCAAGAUAUGCUG
`10nt
`8nt
`6nt
`4nt
`UUACUUACCAAGUUCUAUACG-5'
`4nt
`6nt 8nt 10nt
`(ss/passenger strand) ΔG=-9,1 kcal/mol
`increasing 3’-2’OMe
`modifications
`
`0.1 nM
`1 nM
`Concentration of transfected siRNA
`(as) guide strand labelled
`increasing 3’-2’OMe nucleotides
`
`10 nM
`
`(ss) passenger strand labelled
`increasing 3’-2’OMe nucleotides
`
`B
`
`110
`100
`90
`80
`70
`60
`50
`40
`30
`20
`10
`0
`
`Normalized ratio of RL/FL activity
`
`NegsiRNA
`
`siTK3-as3’4nt/ss3’4nt
`
`siTK3-as3’4nt/ss3’8nt
`
`siTK3-as3’8nt/ss3’4nt
`
`0.1 nM
`1 nM
`10 nM
`Concentration of transfected siRNA
`
`3h
`12h
`4nt-3'-as/ss
`as-Cy5
`
`3h
`12h
`8nt-3'-as/ss
`as-Cy5
`
`3h
`12h
`4nt-3'-as/ss
`ss-Cy5
`
`3h
`12h
`8nt-3'-as/ss
`ss-Cy5
`
`3h
`12h
`8nt-3'-as
`ss-Cy5
`
`3h
`12h
`8nt-3'-ss
`ss-Cy5
`
`40
`
`35
`
`30
`
`25
`
`20
`
`15
`
`10
`
`05
`
`D
`
`EGFP-Ago2 interaction with labelled
`
`strand in %
`
`3h
`12h
`4nt-3'-as/ss
`as-Cy5
`
`3h
`12h
`6nt-3'-as/ss
`as-Cy5
`
`3h
`12h
`8nt-3'-as/ss
`as-Cy5
`
`3h
`12h
`10nt-3'-as/ss
`as-Cy5
`
`3h
`12h
`4nt-3'-as/ss
`ss-Cy5
`
`3h
`12h
`6nt-3'-as/ss
`ss-Cy5
`
`3h
`12h
`8nt-3'-as/ss
`ss-Cy5
`
`3h
`12h
`10nt-3'-as/ss
`ss-Cy5
`
`130
`120
`110
`100
`90
`80
`70
`60
`50
`40
`30
`20
`10
`0
`
`A
`
`Normalized ratio of RL/FL activity
`
`C
`
`40
`
`35
`
`30
`
`25
`
`20
`
`15
`
`10
`
`05
`
`EGFP-Ago2 interaction with labelled
`
`strand in %
`
`0
`0
`-OMe modifications on silencing, guide-strand incorporation, strand separation, and asymmetry. (A) Illustration of the siTK3
`-2
`FIGURE 2 Effects of 3
`0
`OMe nt positions of the various modified strands. ER293 cells were transfected with the indicated amounts of differently modified siTK3
`siRNA with 2
`together with the fixed concentration of the pGL2-Control and pRL-TK reporter plasmids. After 48 h, the ratios of target to control luciferase concentration
`0
`0
`0
`were normalized to the NegsiRNA control (black bar). Unmodified siTK3, lined bar; siTK3 3
`4nt, light gray; siTK3 3
`6nt, gray; siTK3 3
`8nt, dark gray;
`0
`10nt, open bar. The plotted data were averaged from six independent experiments 5 SD. (B) ER293 cells were transfected with the indicated
`siTK3 3
`0
`0
`-end with either 4 or 8 2
`-OMe nt as illustrated in the graph. After 48 h, the ratios of target to control luciferase concen-
`amounts of siTK3 modified on the 3
`tration were normalized to the NegsiRNA control. The plotted data were averaged from six independent experiments 5 SD. (C and D) Cross-correlation
`amplitudes in the cytoplasm (solid bars) and nucleus (open bars) after 3 and 12 h of incubation for the indicated siRNAs. Mean values 5 SE.
`
`0
`
`strand (23) and high levels of Renilla luciferase silencing
`(Fig. 2 A).
`-OMe modifica-
`To investigate the positional effects of 2
`tions within small RNAs, we generated siRNAs with con-
`0
`-OMe modifications on
`stantly increasing numbers of 2
`0
`0
`-end (Fig. 2 A). The introduction of 4 2
`-OMe-modi-
`the 3
`0
`-end resulted in increased silencing
`fied nucleotides at the 3
`activity compared with unmodified siRNAs as detected in
`a dual-luciferase silencing readout. On the other side, the
`0
`-OMe nt resulted in strongly reduced
`introduction of 6 2
`silencing activity and was completely abolished with
`0
`0
`-end (Fig. 2 A). To investigate
`-OMe nt on the 3
`8 and 10 2
`whether the inhibition was caused by modification of the
`guide or passenger strand, we measured the silencing
`activity of only partially modified siRNA duplexes. We de-
`tected identical silencing rates for the siRNA duplex modi-
`0
`-OMe nt compared
`fied on the passenger strand with 8 2
`with the control, whereas the modification on the guide
`strand resulted in an almost complete loss of silencing
`activity (Fig. 2 B). In agreement with the results for the
`0
`-end
`siTK3 duplex, a second siRNA modified on the 3
`0
`-OMe nt targeting Renilla luciferase completely
`with 8 2
`lost its silencing activity (data not shown).
`
`Biophysical Journal 100(12) 2981–2990
`
`To further characterize the mechanism of inhibition, we
`tested the interaction of the differently modified siRNAs
`with Ago2 by FCCS in living cells. To that end, we microin-
`jected guide-strand-labeled siTK3 duplexes into EGFP-
`Ago2 cells and analyzed the amount of loaded Ago2 by
`FCCS after 3 and 12 h (Fig. 2 C). We specifically chose these
`two time points because the 3 h value represents the early
`(still rising RISC-loading level) and 12 h represents the
`late RISC-activation phase (already declining RISC-loading
`level) (28). Therefore, they are ideally suited to investigate
`strand separation and RISC stability, as both time points
`display almost identical loading levels in the cytoplasm.
`Under our experimental conditions with guide-strand-
`labeled siTK3 duplexes, we detected no differences in the
`guide-strand affinity to Ago2 between a silencing active
`4 nt modified duplex and the silencing impaired duplexes
`0
`OMe nt. All labeled guide strands were
`containing 6–10 2
`incorporated equally well with cross-correlation amplitudes
`of ~30–35% (Fig. 2 C). This indicates that the silencing
`inhibition is not a result of reduced affinities to the RLC
`0
`-OMe modification. Therefore,
`the
`resulting from the 2
`0
`0
`-OMe modification on the 3
`-end does not influence the
`2
`affinity to the RLC/RISC.
`
`

`

`Effects of 2
`
`0
`
`-OMe Modified siRNAs
`
`0
`
`-OMe modifications on strand
`To study the effect of 2
`separation and asymmetry, we microinjected the aforemen-
`tioned modified siTK3 duplexes labeled on the passenger
`strand and compared the resulting cross-correlation ampli-
`tudes with the guide-strand-labeled duplexes (Fig. 2 C).
`0
`-OMe modifica-
`The silencing active duplex containing 4 2
`0
`-end displayed almost no interaction of RISC
`tions on the 3
`with the labeled passenger strand. With increasing amounts
`0
`-OMe nt, the interaction of the labeled
`of introduced 2
`passenger strand with RISC increased in the cytoplasm as
`well as in the nucleus. For the completely inhibited duplexes
`0
`-OMe nt, we obtained almost identical
`containing 8 and 10 2
`incorporation levels in the cytoplasm after 3 h compared
`with guide-strand-labeled duplexes, whereas after 12 h we
`detected a clear reduction of RISC-interacting passenger
`strands (Fig. 2 C). Because we detected silencing inhibition
`0
`-OMe modifications only on the guide strand (Fig. 2 B),
`by 2
`we microinjected siRNAs that were modified with 8 nt
`0
`-OMe modifications on the guide or passenger strand
`2
`only, and investigated passenger-strand release after 3 and
`12 h (Fig. 2 D). In agreement with the silencing experi-
`ments, we found that strand separation was mainly inhibited
`when the guide strand of the duplex was modified, whereas
`the modified passenger strand showed only marginal effects
`0
`(Fig. 2 D). These results indicate that 2
`-OMe-modified
`duplexes do not interfere with the asymmetry or the binding
`affinity to RISC, but clearly inhibit the strand separation
`during the activation of RISC. A selection of the measured
`cross-correlation curves is shown in Fig. S4 A.
`We note that the level of nuclear-loaded RISC constantly
`increased with larger amounts of remaining passenger strand
`in RISC after 3 h. A similar effect was previously observed
`for nuclear RISC targeting 7SK snoRNA inhibited in
`target-mediated cleavage by secondary structures in the
`target RNA or bulges between the target guide strand at
`the cleavage site (28). In addition, it has been shown that
`
`2985
`
`hAgo2 containing RISC cleaves the passenger strand during
`activation to facilitate strand separation (30). Therefore, we
`tested the hAgo2-mediated cleavage activity of RISC loaded
`0
`-OMe nt in-
`with a guide strand modified with either 4 or 8 2
`vitro (Fig. 3 A). Consistent with the dual luciferase silencing
`assays, only siTK3-4nt was able to specifically cleave the
`siTK3 target RNA, whereas siTK3-8nt displayed no detect-
`0
`OMe nt on the
`able cleavage activity. Therefore, the 8 2
`0
`-end clearly inhibited Ago2-mediated cleavage, thereby
`3
`interfering with passenger-strand cleavage and release, as
`indicated by the increased levels of passenger strand
`compared with siTK3-4nt after microinjection (Fig. 2 C).
`Of interest, the level of passenger-strand-labeled duplex
`bound to RISC strongly decreased after 12 h compared
`with 3 h. This indicates that in addition to the cleavage-
`mediated passenger-strand release, a bypass mechanism
`exists that also results in strand separation and passenger-
`strand removal (Fig. 2 C). The possibility that the duplex
`was released during the incorporation period can be
`excluded because we did not obtain reduced interaction
`levels of the guide-labeled duplex. This bypass mechanism
`is much slower and time-consuming than the cleavage-medi-
`ated mechanism.
`In a previous study (28) we showed that it is possible to
`investigate RISC-target interactions via FCCS in living cells
`by microinjecting a 50-nt-long, single-stranded, and fluores-
`cently labeled RNA containing the target sequence for
`0
`-OMe nt on both ends.
`siTK3, which is stabilized by 7 2
`FCCS measurements in nontransfected cells and cells trans-
`fected with a control siRNA sharing no sequence comple-
`mentarity with the target RNA (NegsiRNA) displayed no
`interaction between EGFP-Ago2/RISC and the target
`RNA after 1–3 h (Fig. 3 B). This demonstrates the absence
`of miRNA-binding sites within the target RNA and the spec-
`ificity of the RISC-target RNA interaction assay, as the pres-
`ence of siRNAs leading to an miRNA-like interaction
`
`1h
`
`2h
`
`3h
`
`0123456
`
`Ratio target-RNA/EGFP-Ago2
`
`1h
`
`2h
`
`3h
`
`20
`16
`12
`
`048
`
`EGFP-Ago2 particles
`
`1h
`2h
`3h
`No siRNA
`
`1h
`2h
`3h
`1h
`2h
`3h
`1h
`2h
`3h
`NegsiRNA siTK3-bulge siTK3-4nt
`
`1h
`2h
`3h
`siTK3-6nt
`
`1h
`2h
`3h
`siTK3-8nt
`
`1h
`2h
`3h
`siTK3-10nt
`
`18
`16
`14
`12
`10
`
`02468
`
`B
`
` EGFP-Ago2 bound to target-RNA in %
`
`
`
`-target RNA
`
`-5’-cleavage
` product
`
`A
`
`Buffer
`
`Mock
`
`siTK3-4nt
`
`siTK3-8nt
`
`0
`0
`-OMe modifications on target interaction and cleavage. (A) The cleavage activity of siTK3-4nt or -8nt was analyzed in vitro.
`-2
`FIGURE 3 Effects of 3
`Only siTK3-4nt was able to cleave the target RNA; no cleavage product can be observed in buffer only, mock treated cells, or siTK3-8nt. (B) EGFP-Ago2
`serves as a fluorescent label for RISC that can interact with the Cy5-labeled target RNA. The interaction with the target RNA is determined by FCCS in living
`cells. Cross-correlation measurements were performed 1, 2, or 3 h after microinjection. Data are represented as mean 5 SE; solid and open bars indicate
`measurements obtained in the cytoplasm and nucleus, respectively. The number of particles of EGFP-Ago2 and the ratio of target RNA/EGFP-Ago2 for the
`0
`-OMe nt are shown.
`experiments with the siRNAs modified with 4, 6, 8, and 10 2
`
`Biophysical Journal 100(12) 2981–2990
`
`

`

`2986
`
`Ohrt et al.
`
`results in increased levels of target RNA bound to RISC
`(Fig. 3 B). The fraction of bound target RNA by RISC
`was strongly decreased in the presence of a perfect matching
`guide strand (siTK3-4nt) due to the cleavage of the target
`RNA followed by the release of the cleavage products
`from RISC (Fig. 3 B). The levels of RISC bound to the target
`0
`-OMe
`RNA increased with the amount of incorporated 2
`modifications and correlated with the aforementioned
`silencing activities of the siRNAs containing 6–10 nt at
`0
`-end (Fig. 3 B). A selection of the measured cross-
`the 3
`correlation curves is shown in Fig. S4 B. To rule out any
`concentration-mediated effects or changed EGFP-Ago2
`expression levels, the average number of particles obtained
`from the autocorrelation curves is shown in the inset of
`Fig. 3 B, which clearly demonstrates homogeneous levels
`for both species over the time of the experiment. Of interest,
`RISC loaded with the modified guide RNAs containing
`0
`-OMe nt displayed even higher interaction levels
`8–10 2
`compared with RISC loaded with a bulge-forming guide
`strand. These higher amplitudes may result from a stabiliza-
`0
`-OMe modification on the RISC-target
`tion effect of the 2
`interaction. This can be caused by the increased affinity
`due to the complete hybridization of the guide-target RNA
`
`duplex or by the protection of the guide strand against
`nucleases.
`
`0
`
`0
`-end lead to
`-OMe modifications on the 5
`2
`destabilization of the RISC-guide RNA interaction
`0
`
`-OMe modifications on
`After characterizing the effect of 2
`0
`0
`-end, we wanted to test the influence of two 2
`-OMe
`the 3
`0
`-end of siRNAs. In the
`modified nucleotides on the 5
`silencing readouts, a strong decrease of silencing activity
`0
`-OMe modifications were present
`was detected when the 2
`0
`-end (Fig. 4 A). To identify whether the inhibition
`on the 5
`results from the guide or the passenger strand only, we
`also analyzed duplexes with either
`the guide or
`the
`0
`-end guide-strand
`passenger strand. The duplex with the 5
`modification showed the same silencing inhibition as the
`dual-modified siRNA, whereas the modification on the
`passenger strand had no impact on the silencing activity
`0
`(Fig. 4 A). We then analyzed the affinity of the 5
`-end
`modified siRNAs to RISC by microinjecting the guide-
`strand-labeled duplex. We detected the same amount of
`guide-strand-loaded RISC compared with the unmodified
`duplex after 3 h (Fig. 4 B). From this result we can conclude
`
`3h
`12h
`4nt-3'-as/ss
`as-Cy5
`
`3h
`12h
`4nt-3'-as/ss
`ss-Cy5
`
`3h
`12h
`2nt-5'-as/ss
`as-Cy5
`
`3h
`12h
`2nt-5'-as/ss
`ss-Cy5
`
`siTK3-*
`
`*8ntas/ss
`
`*6ntas/ss
`
`*5’2ntas/ss
`
`*4ntas/ss
`
`1h
`
`*10ntas/ss
`2h
`3h
`
`20
`
`40
`
`60
`Knock down in %
`
`80
`
`100
`
`120
`
`40
`
`35
`
`30
`
`25
`
`20
`
`15
`
`10
`
`05
`
`B
`
`EGFP-Ago2 interaction with labelled
`
` strand in %
`
`20
`18
`16
`14
`12
`10
`
`02468
`
`D
`
`EGFP-Ago2 bound to target-RNA in %
`
`1h
`2h
`3h
`NegsiRNA
`0
`0
`-OMe modifications on silencing, guide-strand incorporation, strand separation, asymmetry, and RISC-target interaction. (A)
`-2
`FIGURE 4 Effects of 5
`ER293 cells were transfected with the indicated amounts of differently modified siTK3 together with the fixed concentration of the pGL2-Control and
`pRL-TK reporter plasmids. After 48 h, the ratios of target to control luciferase concentration were normalized to the NegsiRNA control (indicated in black).
`0
`0
`0
`0
`0
`0
`0
`0
`0
`4nt-as/ss, light gray; siTK3-5
`2-3
`4nt-as/ss, lined bar; siTK3-as3
`4nt/ss5
`2-3
`4nt, dark gray; siTK3-as5
`2-3
`4nt/ss3
`4nt, open bar. The plotted data
`siTK3-3
`were averaged from six independent experiments 5 SD. (B) Cross-correlation amplitudes in the cytoplasm (solid bars) and nucleus (open bars) after 3 and
`12 h of incubation for the indicated siRNAs (mean 5 SE). (C) Target interaction levels with EGFP-RISC determined by FCCS in living cells. Cross-corre-
`lation measurements were performed 1, 2, or 3 h after microinjection of labeled target RNA. Data are represented as the mean 5 SE; solid and open bars
`indicate measurements obtained in the cytoplasm and nucleus, respectively. (D) The levels of target RNA bound to EGFP-Ago2 are plotted against the
`silencing values obtained for the siRNAs at 1 nM concentration after 48 h: 1 h, black diamonds; 2 h, open squares; 3 h, open triangles.
`
`NegsiRNA
`siTK3-3'4nt-as/ss
`siTK3-5'2-3'4nt-as/ss
`siTK3-as3'4nt/ss5'2-3'4nt
`siTK3-as5'2-3'4nt/ss3'4nt
`
`0.1 nM
`1 n