7298–7307 Nucleic Acids Research, 2010, Vol. 38, No. 20
`doi:10.1093/nar/gkq621
`
`Published online 12 July 2010
`
`Solution-state structure of a fully alternately
`20-F/20-OMe modified 42-nt dimeric siRNA construct
`
`Peter Podbevsek1, Charles R. Allerson2, Balkrishen Bhat2 and Janez Plavec1,3,4,*
`
`1Slovenian NMR Center, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia,
`2Department of Medicinal Chemistry, Isis Phamaceuticals, Inc., 1896 Rutherford Road, Carlsbad CA 92008,
`USA, 3Faculty of Chemistry and Chemical Technology, University of Ljubljana and 4EN-FIST Center of
`Excellence, SI-1000 Ljubljana, Slovenia
`
`Received May 5, 2010; Revised June 21, 2010; Accepted June 27, 2010
`
`ABSTRACT
`
`A high-resolution solution structure of a stable 42-nt
`RNA dimeric construct has been derived based on a
`high number of NMR observables including nuclear
`overhauser effects (NOEs), J-coupling constants
`and residual dipolar couplings (RDCs), which were
`all obtained with isotopically unlabeled molecules.
`Two 21-nt siRNA that efficiently hybridize consist
`of ribose units that were alternately substituted by
`20-fluoro or 20-methoxy groups. Structure calcula-
`tions utilized a set of H-F RDC values for all
`21 20-fluoro modified nucleotides under conditions
`of weak alignment achieved by Pf1 phages. A com-
`pletely 20-F/20-OMe modified dimeric RNA construct
`adopts an antiparallel double-helical structure con-
`sisting of 19 Watson–Crick base pairs with addition-
`al 30 UU overhangs and a 50 phosphate group on the
`antisense strand. NMR data suggest that the stabil-
`ity of individual base pairs is not uniform throughout
`the construct. While most of the double helical
`segment exhibits well dispersed imino resonances,
`the last three base pairs either display uncharacter-
`istic chemical shifts of imino protons or absence of
`imino resonances even at
`lower
`temperatures.
`Accessibility of imino protons to solvent exchange
`suggests a difference in stability of duplex ends,
`which might be of importance for incorporation of
`the guide siRNA strand into a RISC.
`
`INTRODUCTION
`
`During recent years, it has become evident that small regu-
`latory RNAs play an important role in many biological
`processes including control of mechanisms directing gene
`
`expression and thus can be exploited for various thera-
`peutic purposes. An accidental discovery, which suggested
`that RNA can be used for silencing virtually any gene, has
`opened up a new field of gene therapy (1–5). The mech-
`anism, termed RNA interference (RNAi), is triggered by
`short RNA duplexes (6–8). Naturally occurring siRNAs
`are produced by enzymatic cleavage of a longer double
`stranded RNA molecule into shorter RNA duplexes.
`siRNAs are typically 21-nt long and contain 2-nt 30 over-
`hangs and 50 phosphates. Once in cells, siRNA associates
`with
`argonaute
`and
`other
`proteins
`forming
`an
`RNA-induced silencing complex (RISC) (9). One strand
`of the siRNA duplex is degraded, while the other strand
`stays bound to the RISC and base pairs with a messenger
`RNA (mRNA) containing the complementary sequence.
`The proteins within RISC possess nuclease activity and
`cleave the target mRNA at a single site. Binding of
`RNA to a complementary region on its target mRNA
`leads to gene silencing by translational repression and/or
`mRNA degradation. This reduces the level of target
`mRNA in cells and effectively knocks down a specific
`gene.
`Since siRNAs can be artificially introduced into cells by
`various methods, RNAi has become a powerful tool for
`the regulation of gene expression. Unfortunately, the
`initial promises of the use of synthetic siRNAs consisting
`of solely standard nucleotides proved problematic. One of
`the main difficulties is the short half-life of unmodified
`RNA in serum due to the activity of endo- and exonucle-
`ases. In order to overcome the problem of instability
`against nuclease degradation, a variety of chemically
`modified nucleotides have been utilized in synthetic
`siRNAs (10–12). Earlier studies showed that the 20-OH
`group is not required for siRNAs to elicit an RNAi
`response (13). Therefore, the 20 position on the sugar
`moiety has been extensively modified. It has been shown
`that 20-O-methyl (20-OMe) (14) and 20-deoxy-20-fluoro
`
`*To whom correspondence should be addressed. Tel: +386 1 4760353; Fax: +386 1 4760300; Email: janez.plavec@ki.si
`Present address:
`Balkrishen Bhat, Regulus Therapeutics, 1896 Rutherford Road, Carlsbad CA 92008, USA.
`
`ß The Author(s) 2010. Published by Oxford University Press.
`This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
`by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
`
`

`

`(20-F) (15) modifications (Figure 1) increase the nuclease
`stability of siRNAs, and at the same time retain its RNAi
`activity. Second generation antisense oligonucleotides
`incorporating these 20-modifications exhibit high binding
`affinity to the target RNA, enhanced metabolic stability,
`and improved pharmacokinetic and toxicity profiles (16).
`Several reports have discussed different extents of substi-
`tution with modified nucleotides ranging from just a single
`nucleotide to the use of uniform modification along both
`strands (12).
`Recently, Bhat and coworkers identified a highly potent
`siRNA duplex, which targets the B site of the human
`phosphatase and tensin homolog mRNA, which was pre-
`viously shown to be a target for degradation using the
`siRNA approach (17). The fully modified duplex, which
`is comprised of alternating 20-F and 20-OMe nucleotides
`exhibits several desirable pharmacokinetic properties (18).
`The melting temperature of the modified duplex is 20C
`higher in comparison to an unmodified RNA oligonucleo-
`tide. High thermal stability is, at least in part, responsible
`for the significantly higher plasma stability observed in the
`modified siRNA. Consequently, 20-F/20-OMe siRNA
`showed a more than a 500-fold increase in in vitro
`potency versus unmodified siRNA (18,19).
`20-modifications can influence the sugar conformation
`to adopt a predominantly North-type (N) sugar pucker,
`and can also improve properties such as affinity (20–24).
`20-modifications that provide a gauche effect and/or a
`charge effect can play a significant role in the level of
`nuclease resistance. Interplay of stereoelectronic effects,
`sequence, hydration or metal ion interactions can influ-
`ence equilibrium between canonical A- and B-type
`duplex and other conformations.
`The focus of the current study is on a 42-nt dimeric
`RNA construct which consists of 19 Watson–Crick base
`pairs, 30 UU overhangs in both strands, which are found
`
`Figure 1. Schematic representation of 20-deoxy-20-fluoro (left) and
`20-OMe (right) substituted ribose units in the preferred N-type sugar
`conformation.
`
`Nucleic Acids Research, 2010, Vol. 38, No. 20 7299
`
`in naturally occurring siRNAs, and a 50 phosphate on the
`antisense strand, which is required for the correct pos-
`itioning within the RISC (Figure 2). We attempt to
`analyze conformational preorganization of the two 21-nt
`RNAs separately and after hybridization into the dimeric
`construct by solution state NMR without the use of 13C
`and 15N isotope labeling. Structural characterization of
`the dimeric construct provides molecular details which
`correlate with the high potency of the modified siRNA.
`Overall topology and details of the 3D structure of the
`fully 20-F/20-OMe modified siRNA duplex together with
`the dynamics of individual segments offer deeper insights
`into structural features as well as the role of 30 overhangs.
`At the outset, we expected to observe differences in duplex
`stability that would be localized to the specific section of
`the construct. Contiguous base pairs in the 50 seed region
`and near the cleavage site are important for nuclease
`activity (25,26). Disruption of
`the helical duplex
`geometry may be important for intermolecular inter-
`actions of a RNA complex with its complementary
`region on a target mRNA which is at the heart of gene
`silencing by translational repression or degradation.
`
`MATERIALS AND METHODS
`
`Sample preparation and NMR data collection
`
`The NMR sample was prepared by dissolving the oligo-
`nucleotides ISIS 401156 (50-GGGUAAAUACAUUCUU
`CAUUU-30) and ISIS 401157 (P-50-AUGAAGAAUGUA
`UUUACCCUU-30) in a 100% 2H2O or 5% 2H2O, 95%
`H2O aqueous solution containing 20 mM NaCl and
`20 mM sodium
`phosphate
`buffer
`(pH = 6.8).
`Oligonucleotide concentration in the NMR sample was
`2.0 mM per strand. The sample used to measure residual
`dipolar couplings (RDCs) was prepared by redissolving
`the NMR sample in a 100% 2H2O filamentous Pf1
`phage solution (Asla Ltd). The total phage concentration
`was 17 mg/ml which resulted in a deuterium splitting of
`17.1 Hz at 800 MHz. 1D 1H and 2D NOESY, DQF-COSY
`and TOCSY NMR spectra were recorded on Varian
`VNMRS 800 MHz spectrometer equipped with a cold
`probe. DPFGSE water suppression scheme was used for
`suppression of the water signal. 2D HP-COSY spectra
`were recorded on a Varian VNMRS 600 MHz spectrom-
`eter with a penta probe. All experiments were performed
`on a natural abundance sample at temperatures ranging
`from 0 to 35C. NMR spectra were processed and
`
`Figure 2. Base pairing pattern of the 20-F/20-OMe modified sense/passenger and antisense/guide RNA strands. 20-F and 20-OMe modified nucleotides
`are depicted in green and blue, respectively. The base paired segments of the strands that displayed observable imino resonances are shaded in red.
`
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`

`7300 Nucleic Acids Research, 2010, Vol. 38, No. 20
`
`analyzed using VNMRJ (Varian Inc.) and Sparky
`software (UCSF).
`
`No other deviations in the two final families of structures
`could be observed.
`
`Tm measurement
`
`A solution for measuring the thermal stability of the
`fully-modified duplex was prepared by combining equal
`volumes of 8 mM solutions of each oligonucleotide in a
`buffer of 100 mM NaCl, 10 mM sodium phosphate
`(pH 7) and 0.1 M EDTA, yielding final concentration
`of 4 mM duplex. A Cary 100 Bio spectrophotometer with
`the Cary Win UV Thermal program was used to measure
`absorbance versus temperature. The solution was heated
`at 0.5C/min from 15 to 95C (repeated in triplicate), and
`Tm was determined from the A260 versus temperature
`curve.
`
`Structure calculations
`
`Structure calculations were performed using AMBER 9
`software with the parmbsc0 force field (27). Force field
`parameters for the modified nucleotides were used from
`previous studies (28,29). Initial starting structure was
`created using the InsightII software. The structure was
`then subjected to 100 ps of NMR restrained simulated an-
`nealing (SA) calculations using a generalized Born implicit
`solvation model. For each SA calculation a random
`starting velocity was used. For the first 15 ms the mol-
`ecules were held at a constant temperature of 300 K. The
`molecules were then heated to 1000 K in the next 10 ps,
`after which temperature was constant for 25 ps, scaled
`down to 100 K in the next 25 ps and reduced to 0 K in
`the
`last
`25 ps.
`The
`force
`constants
`were
`1 A˚ 2 for nuclear overhauser effect (NOE)
`35 kcal mol
`2
`1 rad
`for
`torsion angle and
`distance, 300 kcal mol
`1 A˚ 2 for base planarity restraints. Direct
`25 kcal mol
`dipolar HF coupling restraints were used to define the
`five unique elements of the alignment tensor. In the
`AMBER software, these elements are treated as additional
`variables and are optimized along with the structural par-
`ameters. The variables used are the Cartesian components
`of the alignment tensor in the axis system defined by the
`molecule itself. The alignment tensor was fitted to the
`starting structure, and the tensor obtained from this
`fitting was used as the initial guess for further refinement.
`The cutoff for non-bonded interactions was 20 A˚ . The
`SHAKE algorithm for hydrogen atoms was used with a
`tolerance of 0.0005 A˚ . All structures from SA were sub-
`jected to a maximum of 10 000 steps of steepest descent
`minimization. A family of 10 minimized structures with
`the lowest energy and the smallest NMR violations were
`selected for further analysis. Helical parameters were
`determined with 3DNA 2.0 software (30). We have also
`performed the structural calculations without the use of
`base planarity restraints. The convergence and overall
`RMSD of the final structures were comparable to the
`original set of structures. Analysis of local base-pair par-
`ameters showed that the average propeller twist changed
`from 6.7 (SD 0.6, maximum deviation per base pair 3.7)
`to 10.1 (SD 1.2, maximum deviation per base pair 7.0)
`with and without base planarity restraints, respectively.
`
`Coordinate deposition
`
`The coordinates for the family of the 10 lowest energy
`structures of the 20-F/20-OMe modified dimeric 42-nt con-
`struct have been deposited in the Protein Data Bank with
`the accession code 2KWG.
`
`RESULTS
`
`Single-strand preorganization
`Solutions of individual 20-F/20-OMe modified 21-nt RNA
`oligonucleotides were prepared in 5% 2H2O, 95% H2O
`and transferred into NMR tubes. 1D 1H NMR spectrum
`of the sense strand exhibits three imino resonances at 15C
`(Figure 3A and B). The resonance at d12.4 p.p.m. reveals a
`slightly broader width at half-height in comparison to the
`remaining two resonances and undergoes
`significant
`broadening upon lowering the temperature to 0C. The
`chemical
`shifts of
`the two sharp imino resonances
`suggest the formation of one AU and one GC base pair.
`Imino–imino cross-peak in NOESY spectrum (data not
`shown) indicates that the two base pairs are adjacent.
`The base pairing pattern can be associated with one of
`the topologies suggested by the MFOLD program (31)
`(Figure 3A). No NOESY cross-peaks at d12.4 p.p.m.
`could be observed due to the broad nature of resonance
`signal.
`Five imino resonances in the range from d11.0 to
`14.0 p.p.m. could be observed in the 1D 1H NMR
`spectrum of the antisense strand at 15C (Figure 3C and
`D). On the basis of chemical shifts, the two upfield imino
`resonances were assigned to a GU base pair, which was
`confirmed by the strong NOESY imino–imino cross-peak
`between them. The GU base pair is adjacent to an AU
`base pair with its imino proton resonating at d14.0 p.p.m.
`The two remaining resonances at d13.1 and 13.2 p.p.m.
`exhibit a NOESY cross-peak between each other, but no
`cross-peaks to other imino protons. The resonances at
`d13.1 and 13.2 p.p.m. correspond to adjacent AU and
`GC base pairs, respectively. The base pairing pattern
`could be linked to one of the topologies suggested by
`the MFOLD program (31) (Figure 3C).
`
`Dimeric construct exhibits well resolved NMR signals
`Equimolar amounts of the two 20-F/20-OMe modified
`21-nt RNA oligonucleotides in 5% 2H2O, 95% H2O
`were transferred into an NMR tube. 1D 1H 800 MHz
`NMR spectrum exhibited sharp and well resolved reson-
`ances (Figure 4). Seventeen imino resonances could be
`identified in the region from d11.5 to 14.2 p.p.m.
`indicating Watson–Crick base pairing, which suggests
`the formation of a single double helical structure. A
`second sample of equimolar amounts of the 20-F/20-OMe
`oligonucleotides was prepared in phosphate buffer
`solution, and was used to measure the thermal hybridiza-
`tion stability (Tm) of
`the resulting duplex. A single
`81.7C,
`hyperchromic
`transition was observed at
`
`

`

`Nucleic Acids Research, 2010, Vol. 38, No. 20 7301
`
`Figure 3. Base pairing pattern of the sense (A) and antisense (C) strands and the corresponding 1D 1H NMR spectra of individual sense (B) and
`antisense (D) strands recorded at 15C.
`
`Figure 4. 1D 1H NMR spectrum of the dimeric RNA construct recorded at 25C. Imino region with the assignment of resonances is shown in the
`inset.
`
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`

`7302 Nucleic Acids Research, 2010, Vol. 38, No. 20
`
`confirming the formation of a single base paired species.
`Detailed temperature-dependent NMR measurements
`were used to assess the local thermal stability of individual
`base pairs. The NMR melting profiles in temperature
`range from 25 to 80C reveal that the melting of the
`duplex is not uniform and that base pairs located in the
`labile segment of the helix exhibit noticeably lower melting
`temperatures in comparison to the rest of the construct
`(Supplementary Figure S1). U15, U16 and G24 imino
`proton resonances which are resolved throughout the tem-
`perature range can be observed at temperatures of up to
`55C. On the other hand, G27 imino proton, which con-
`stitutes the next base pair is observed at temperatures of
`up to 70C. Other imino resonances cannot be uniquely
`identified due to distinct temperature variation of their
`chemical shift and severe spectral overlap. They can be
`temperatures of up to 65C.
`observed, however, at
`Interestingly, some imino peaks are observed even at
`80C.
`the imino resonances was possible
`Assignment of
`through the imino–imino sequential walk in 2D NOESY
`spectra (Figure 5). The imino peaks indicated the forma-
`tion of continuous Watson–Crick base pairs between
`G1-C40 and U16-A25 pairs. The additional C17 G24
`base pair was inferred through the observation of G24
`imino proton at the chemical shift of d11.5 p.p.m. This
`chemical shift of a GC Watson–Crick base pair suggests
`specific stacking interactions or other structural features at
`the end of a perfectly hydrogen bonded double helix. The
`imino proton of a GC base pair flanked by a UA base pair
`at the 50 end and an AU base pair at the 30 end could be
`shielded by over 2 p.p.m. in an A-RNA helix (32). It is
`noteworthy, that the signal intensity and the half-width of
`the G24 imino proton is comparable to other imino
`signals. No imino proton resonances could be detected
`
`the 2D NOESY spectrum
`Figure 5. The imino–imino region of
`(tm = 250 ms, 15C). Lines represent the sequential walk for the base
`paired region (G1-C17 and G24-C40).
`
`for hypothetical A18 U23 and U19 A22 base pairs at tem-
`peratures as low as 0C.
`
`Assignment of resonances
`
`Assignment of imino protons, which established second-
`ary structure was followed by more detailed assignment of
`non-exchangeable protons (33,34). Pyrimidine H5 protons
`were identified with the use of 2D DQF-COSY spectra
`showing through bond correlations with aromatic H6
`protons of the same residue. Cytosine H5 protons ex-
`hibited strong intranucleotide NOESY correlations with
`amino protons, which in turn showed NOE cross-peaks to
`base paired guanine imino protons. Adenine H2 reson-
`ances were assigned through their strong NOE correl-
`ations with base paired uracil imino protons. Sequential
`aromatic (H6/H8)-sugar H10 connectivities could be
`traced through the whole length of both sense and anti-
`sense strands (Figure 6), which enabled us to assign all
`aromatic and anomeric protons. However, this was done
`with the help of HF couplings which could be observed in
`the 2D NOESY spectra. Specifically, scalar couplings of F
`with H10 helped us to identify sugar protons belonging to
`nucleotides with the 20-F modification. H20 protons of all
`20-F substituted nucleotides and cca 50% of the H30 res-
`onances of 20-F substituted nucleotides could be assigned
`due to their coupling with fluorine nuclei. Only a few H20
`protons of 20-OMe substituted nucleotides could be
`assigned. 20-OMe proton resonances (in the region from
`d3.3 to 3.9 p.p.m.) were assigned by their intra- and
`internucleotide correlations with aromatic protons. 2D
`DQF-COSY spectrum showed no H10-H20 cross-peaks
`indicating that the sugar conformations of all nucleotides
`1H
`adopted predominantly N-type puckering. The
`chemical
`shift values have been deposited in the
`Biological Magnetic Resonance data Bank (BMRB, acces-
`sion no. 16852).
`
`Restraints and structure calculations
`
`NOE distance restraints for non-exchangeable protons
`were obtained from 2D NOESY spectra recorded at
`25C in 100% 2H2O with mixing times ranging from 80
`to 250 ms. The volume of
`the pyrimidine H5-H6
`cross-peak was used as the distance reference (2.45 A˚ ).
`NOE distance restraints for exchangeable protons were
`obtained from 2D DPFGSE NOESY spectra recorded
`at 25C in 5% 2H2O, 95% H2O with mixing time of
`250 ms. Cross-peaks were classified as strong (1.8–3.6 A˚ ),
`medium (2.6–5.0 A˚ ) and weak (3.5–6.5 A˚ ).
`Torsion angle restraints for a and z were set to 0 ± 120
`to exclude trans conformations inferred from the narrow
`range of 31P resonances. Torsion angles " were restrained
`to 235 ± 65 to exclude the unfavorable gauche+ conform-
`ation. Torsion angles b were restrained to 180 ± 40
`(trans) due to the lack of P-H50 and P-H500 cross-peaks
`in HP-COSY spectrum. The backbone torsion angle re-
`straints (a, b, " and z) were used only for the base paired
`region (G1-C17, G24-C40). Due to the absence of H10-H20
`cross-peaks in 2D DQF-COSY spectra, which indicate an
`N-type sugar pucker, torsion angles d of all nucleotides
`were restrained to 85 ± 30. All torsion angles  were
`
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`

`Nucleic Acids Research, 2010, Vol. 38, No. 20 7303
`
`Figure 6. The aromatic–anomeric region of the 2D NOESY spectrum (tm = 150 ms, 25C). Lines represent the sequential walk for the sense,
`G1-U21 (A) and antisense, A22-U42 (B) strands, which are shown separately to reduce overlap.
`
`restrained to 120 ± 90 (anti) based on the intensity of
`intraresidual H6/H8-H10 NOESY cross-peaks.
`The above NOE distance and torsion angle restraints
`were complemented by the implementation of HF residual
`dipolar coupling constants (35). The fluorine nucleus
`(placed on every other nucleotide) is coupled to H10, H20
`and H30 sugar protons. Coupling constants were obtained
`by measuring the HF splittings, which are observable in
`traces of H10-H20 and H20-aromatic cross-peaks
`in
`NOESY spectra. Two bond F-H20 splittings are in the
`range from 42.8 to 53.8 Hz and three bond F-H10 splittings
`are between 12.1 and 18.8 Hz. After the addition of the Pf1
`phages, the splittings were measured again and the RDC
`values were calculated. We were able to collect F-H10 and
`F-H20 RDC values for all 21 nt, which are 20-F modified.
`Measured RDC vales range from 0.7 to 5.2 Hz for F-H20
`
`and from 0.1 to 3.0 Hz for F-H10 coupled pair. F-H30
`splittings were observed in NOESY spectra and are in the
`range from 25 to 30 Hz. However, F-H30 RDCs could not
`be determined with sufficient accuracy due to the unreli-
`the F-H30 splitting and severe
`able measurement of
`spectral overlap which limited assignment of H30 protons.
`Finally, 311 NOE restraints, 219 torsion angles and 42
`RDCs were used in the structure calculation process
`(Table 1). Forty hydrogen bond and 34 bp planarity re-
`straints were used for the 17 AU and GC base pairs. No
`hydrogen bond and planarity restraints were used for the
`hypothetical A18 U23 and U19 A22 base pairs, for which
`no imino signals could be observed.
`A set of 100 structures was calculated using SA protocol
`and energy minimization, from which the 10 lowest energy
`structures were selected. The final set of 10 structures
`
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`7304 Nucleic Acids Research, 2010, Vol. 38, No. 20
`
`Table 1. Structural statistics
`
`NOE-derived distance restraints
`Intranucleotide NOEs
`Sequential NOEs
`Long-range NOEs
`Torsion angle restraints
`Hydrogen bond restraints
`Base pair planarity restraints
`Residual dipolar couplings
`NOE violations >0.3 A˚
`Deviations from idealized covalent geometry
`Bonds (A˚ )
`Angles ()
`Pairwise heavy atom RMSD (A˚ )
`Overall
`Without UU overhangs (1–19, 22–40)
`Only the base paired region (1–17, 24–40)
`
`135
`151
`25
`219
`40
`34
`42
`0
`
`0.010 ± 0.000
`2.48 ± 0.02
`
`2.29
`1.78
`1.54
`
`Figure 7. (A) The lowest energy structure of the 20-F/20-OMe modified
`dimeric 42-nt RNA construct. Nucleotides G1-U16 and A25-C40 are
`depicted in blue, base pair C17 G24 is in yellow and base pairs A18
`U23 and U19 A22 are in orange. (B) Superposition of the 10 lowest
`energy structures of the 42-nt construct. Sense and antisense strands are
`in blue and red, respectively.
`
`exhibits a pairwise heavy atom RMSD of 2.3 A˚ , while the
`base paired region (G1-C17, G24-C40) alone displays a
`RMSD of 1.5 A˚ . The construct adopts an A-type double
`helical structure (Figure 7) with an average rise and twist
`parameters of 2.9 A˚ and 30.5, respectively. For compari-
`son, the rise and twist parameters in standard A-type
`RNA are 2.8 A˚ and 32.8, respectively (36).
`
`Figure 8. Superposition of the 10 lowest energy structures of the U20,
`U21 overhang over the U19 A22 base pair.
`
`Perusal of the structures shown in Figure 7 shows the
`formation of 19 Watson–Crick base pairs in the stem
`region (G1-U19 and A22-C40).
`Interestingly,
`imino
`proton signals of A18-U23 and U19-A22 base pairs have
`not been observed experimentally. In accordance, the
`structure of these two terminal base pairs is less well
`defined, due to the lack of a hydrogen bond, base
`planarity and backbone torsion angle restraints. All
`20-OMe groups in the stem region are rotated towards
`the minor groove. The structure of the U20 and U21
`overhang in the sense strand is not well defined.
`However,
`in all 10 final structures U20 stacks on the
`terminal A19 U22 base pair and in several structures
`U21 stacks on U20 (Figure 8). On the other hand, the
`U41 and U42 overhang in the antisense strand does not
`stack on the terminal G1 C40 base pair, instead, 20-OMe
`group of C40 is positioned above its pyrimidine ring and
`blocks stacking of U41. As a result, the U41 and U42
`overhang is poorly defined and does not adopt a well
`defined structure.
`
`DISCUSSION
`
`Short synthetic oligonucleotides can be used to specifically
`modulate gene expression through the RNA interference
`(RNAi) mechanism. However, the use of such molecules
`in vivo did not bring expected results, partly due to the low
`nuclease stability of siRNA oligonucleotides originating
`from natural nucleotides. Several chemical modifications
`on
`the
`ribose
`ring were
`shown
`to
`improve
`pharmacokinetic properties of synthetic oligonucleotides
`without causing toxicity, which is crucial for delivery of
`such molecules into living organisms. A completely 20-F/
`20-OMe modified dimeric RNA construct was previously
`shown to be a highly potent RNAi trigger in HeLa cells
`(18). In the current study, we present a 3D structure of a
`42-nt dimeric alternately 20-F/20-OMe modified construct
`consisting of a Watson–Crick base paired duplex with
`additional 30 UU overhangs and a 50 phosphate group
`on the antisense strand, which mimics naturally occurring
`siRNAs produced in cells by the Dicer enzyme.
`The two 21-nt oligos efficiently hybridize thus forming
`an A-type double helix with 30 UU overhangs on both
`strands. The helical segment is completely complementary
`
`

`

`and exhibits 19 Watson–Crick base pairs. The overall
`thermal stability of the duplex, reflected by a Tm of
`81.7C, is not appreciably different from the stability of
`an identical duplex lacking the 30 UU overhangs, with a
`previously reported Tm of 82.0C (18). However, NMR
`data suggests that the stability of individual base pairs is
`not uniform throughout the entire length of the construct.
`Most of the double helix (G1-U16, A25-C40), apart from
`the last three base pairs, exhibits well dispersed imino res-
`onances in the Watson–Crick region of NMR spectra.
`However,
`the last 3 bp display somewhat different
`properties. G24 imino proton exhibits a chemical shift of
`d11.5 p.p.m., which is uncharacteristic for imino protons
`involved in GC Watson–Crick base pairs. Interestingly,
`the line-width of
`this signal
`is comparable to other
`imino resonances in the Watson–Crick region. U19 and
`U23 imino resonances involved in AU base pairs could
`not be observed in NMR spectra even after lowering the
`temperature to 0C, which clearly indicates that these
`imino protons are accessible
`to solvent
`exchange.
`Differences in stability of helix segments can be attributed
`to the base pair composition of the construct, which ends
`with two AU base pairs on one end and with three GC
`base pairs on the opposite end. Our findings are in agree-
`ment with previous reports, which suggest that a differ-
`ence in stability of duplex ends is required for the
`incorporation of the correct siRNA strand into RISC
`(37,38).
`During the RISC assembly process, the RISC-loading
`complex loads the siRNA duplex into Ago2 (39). The 50
`end of the guide strand is inserted into the phosphate
`binding pocket of the Ago2 PIWI domain and serves as
`a template for target mRNA recognition. The passenger
`strand is subsequently cleaved by Ago2 and ejected from
`the RISC complex. Guide and passenger strands are
`determined on the basis of relative stabilities of helix
`ends (37,38). The helix end that is less stable interacts
`with the PIWI domain of Ago2, which results in the un-
`winding of the first base pair (40,41). The strand whose 50
`end is at the less stable end of the helix becomes the guide
`strand, while the other strand becomes the passenger
`strand. Our data show a clear difference in the relative
`stability of helix ends. The labile base pairs A18 U23
`and U19 A22 suggest that the A22–U42 strand will serve
`as a guide strand and will thus control the incorporation
`of the siRNA duplex into the RISC complex.
`Although one end of the helix is less stable, the existence
`of fraying ends in solution can be dismissed due to the
`presence of
`several
`inter-strand NOE cross-peaks
`correlating aromatic and sugar protons between U20
`and A22 (Supplementary Figure S2). All
`structures
`exhibit A18 U23 and
`obtained by SA simulations
`U19 A22 base pairs, despite the absence of hydrogen
`bond restraints for these base pairs. Stabilization of
`these base pairs is probably achieved through favorable
`stacking interactions. Furthermore, the U20 and U21
`overhang in the sense strand stacks efficiently on the
`terminal U19 A22 base pair.
`On the other hand, there is poor, if any, base–base
`stacking of the U41 and U42 overhang on the terminal
`GC base pair in the antisense strand. The stacking of U41
`
`Nucleic Acids Research, 2010, Vol. 38, No. 20 7305
`
`is prevented by the 20-OMe group of C40, which is pos-
`itioned directly above the aromatic ring of C40. As a
`result, the structure of the U41, U42 overhang is poorly
`defined. This appears to have no effect on the terminal GC
`base pair. Apart from the G1 imino proton signal being
`slightly broader at 25C, which is expected due to its
`exposure to exchange processes at the helix end, there is
`no indication of exchange of hydrogen bonded imino
`protons with solvent in the rest of the helix (G1-U16,
`A25-C40).
`For comparison, preliminary measurements on the
`native isosequential siRNA were performed. The native
`analogue displays similar features to the 20-F/20-OMe
`modified siRNA. Very good dispersion of the imino
`proton
`chemical
`shifts
`has
`been
`observed
`(Supplementary Figures S3 and S4). As expected, the
`G24 imino proton exhibits an upfield chemical shift due
`to its position between UA and AU base pairs. However,
`a large part of the aromatic and anomeric proton reson-
`ances is condensed in highly overlapped regions, which
`precludes further analysis with an isotopically unlabeled
`RNA sample of this molecular weight (Supplementary
`Figure S5). It is noteworthy that DQF-COSY spectrum
`showed no cross-peaks in H10-H20 region, which is indica-
`tive of N-type sugar conformation.
`All nucleotides in the construct exhibit a predominantly
`N-type sugar pucker, which is usually found in RNA mol-
`ecules. This is partly due to the presence of 20 modifica-
`tions, which favor the C30-endo conformation of the sugar
`ring (20–22). The RNA like
`conformation greatly
`improves the binding affinity for the target RNA, which
`is desired for synthetic siRNA oligonucleotides. It is not
`surprising that the introduction of 20-fluoro and 20-OMe
`modifications does not have any significant impact on the
`structure of the construct. Deviations from the native
`A-type RNA structure could reduce the efficiency of the
`RNAi mechanism. The alternating 20-F/20-OMe motif
`appears to be a promising design for synthetic siRNA
`oligonucleotides with high serum stability.
`
`CONCLUSION
`
`A high-resolution structure of the stable 42-nt RNA
`dimeric construct has been derived based on a high
`number
`of NMR observables
`including NOEs,
`J-coupling constants and RDCs. It is amazing that suffi-
`cient resolution of NMR signals was achieved on an
`800 MHz NMR spectrometer which enabled sequential
`assignment of a 14 kDa dimer without isotope labeling.
`The two 21-nt oligos efficiently hybridized thus forming
`an A-type double helix with 30 UU overhangs on both
`strands. Ribose units in siRNA construct were alternately
`substituted by a 20-deoxy-20-fluoro or 20-methoxy groups
`which contributed to the stabilization of the N-type sugar
`conformation that is typically associated with structure of
`A-form of RNA. Structure calculations utilized a set of
`H-F RDC values under conditions of weak alignment
`achieved by Pf1 phages for all 21 nt, which were 20-F
`modified. A completely 20-F/20-OMe modified dimeric
`RNA construct adopts an