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Human Dicer Binds Short Single-strand and Double-strandRNA with High Affinity and Interacts with Different Regionsof the Nucleic Acids*

Received for publication, May 15, 2008, and in revised form, November 17, 2008 Published, JBC Papers in Press, November 18, 2008, DOI 10.1074/jbc.M803748200

Walt F. Lima1, Heather Murray, Josh G. Nichols, Hongjiang Wu, Hong Sun, Thazha P. Prakash, Andres R. Berdeja,Hans J. Gaus, and Stanley T. CrookeFrom the Department of Molecular and Structural Biology, Isis Pharmaceuticals, Carlsbad, California 92008

Human Dicer is an integral component of the RNA interfer-ence pathway. Dicer processes premicro-RNA and double-strand RNA to, respectively, mature micro-RNA and shortinterfering RNA (siRNA) and transfers the processed productsto the RNA-induced silencing complex. To better understandthe factors that are important for the binding, translocation, andselective recognition of the siRNA strands, we determined thebinding affinities of human Dicer for processed products(siRNA) and short single-strand RNAs (ssRNA). siRNAs andssRNAs competitively inhibited human Dicer activity, suggest-ing that they are interacting with the active site of the enzyme.The dissociation constants (Kd) for unmodified siRNAs were5–11-foldweaker comparedwith a 27-nucleotide double-strandRNA substrate. Chemically modified siRNAs exhibited bindingaffinities for Dicer comparable with the substrate. 3�-Dinucle-otide overhangs in the siRNA affected the binding affinity ofhumanDicer for the siRNAandbiased strand loading intoRNA-induced silencing complex. The Kd values for the ssRNAsranged from 3- to 40-fold weaker than the Kd for the substrate.Sequence composition of the 3�-terminal nucleotides of thessRNAs exhibited the greatest effect on Dicer binding. Dicercleaved substrates containing short siRNA-like double-strandregions and extended 3� or 5� ssRNA overhangs in the adjacentssRNA regions. Remarkably, cleavage sites were observed con-sistent with the enzyme entering the substrate from theextended 3� ssRNA terminus. These data suggest that thesiRNAs and ssRNAs interact predominantly with the PAZdomain of the enzyme. Finally, the tightest binding siRNAswerealso more potent inhibitors of gene expression.

Post-transcriptional gene silencing by the mechanismknown as RNA interference involves the processing of longdouble-strand RNAs (dsRNA)2 into short effector dsRNAs bythe RNase III enzyme, Dicer (1, 2). Human cells contain a single

Dicer isoform, which has been shown to generate both shortinterfering RNAs (siRNA) and mature micro-RNA products(3–6). siRNA and micro-RNA consist of sense and antisensestrands relative to the target mRNA. The siRNA and micro-RNA products of Dicer are loaded into the RNA-inducedsilencing complex (RISC), resulting in either the translationalrepression or destruction of the mRNA target (7–9).Dicer belongs to the RNase III family of enzymes and appears

to be ubiquitous in eukaryotes. The enzyme is a 220-kDa pro-tein consisting of a dsRNA-binding domain, a PAZdomain, twoRNase III domains, and a domain homologous toATP-depend-ent RNA helicase (10–13). The two RNase III domains of Dicerforman intramolecular dimer that together cleave the opposingstrands of the dsRNA, generating 2-nucleotide-long 3�-over-hangs (13). The crystal structure of Dicer from Giardia showedthat the PAZ domain of the enzyme exhibited a tertiary struc-ture similar to the PAZ domain of human Argonaute2 (14). Inaddition, the hydrophobic pocket of the PAZ domain wasresponsible for binding the 3�-dinucleotide overhang of thesubstrate (14). The PAZ and RNase III domains are separatedby an�-helix that functions as amolecular ruler to generate the�20-base pair dsRNA products (14). Finally, the role of theATPase/RNA helicase domain of the enzyme is unclear, sinceno helicase activity has been observed for the enzyme (10).Dicer is a key component of the RISC loading complex (RLC)

(2–5, 15). The minimum RLC consists of human Dicer and thehuman immunodeficiency virus transactivating responseRNA-binding protein (TRBP) (16, 17). The role of the RLC inthe activities of siRNAs is unclear. Several studies showed thatthe reduction of either human Dicer or TRBP protein expres-sion reduced the activities of siRNAs (17, 18). Other studiesshowed that a reduction in Dicer protein expression appearedto have no effect on siRNA activity (19, 20). In addition, syn-thetic Dicer substrates ranging from 25 to 30 nucleotides wereshown to bemore potent inhibitors of gene expression than thecorresponding 21-nucleotide siRNAs, further suggesting a rolefor human Dicer in the activities of siRNAs (21). InDrosophila,cross-linking studies between Dicer-2 and the TRBP paralogR2D2 bound to the siRNA indicated that Dicer-2 was posi-tioned adjacent to the 5� terminus of the sense strand, andR2D2 was positioned adjacent the 5� terminus of the antisensestrand (22). The directionality of the interaction appeared to beinfluenced by the base-pairing stabilities of the siRNA termini(22). Similarly, substrates containing 3�-overhangs were deter-mined to affect the directionality of human Dicer cleavage as

* The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom correspondence should be addressed: Dept. of Molecular andStructural Biology, Isis Pharmaceuticals, 1896 Rutherford Rd., Carlsbad, CA.Tel.: 760-603-2387; Fax: 760-931-0209; E-mail: [email protected].

2 The abbreviations used are: dsRNA, double-strand RNA; siRNA, small inter-fering RNA; ssRNA, single-strand RNA; RISC, RNA-induced silencing com-plex; RLC, RISC loading complex; TRBP, transactivating response RNA-bind-ing protein; GST, glutathione S-transferase; HPLC, high pressure liquidchromatography; 3�-32PCp, 32P-labeled cytidine bisphosphate; pCp, unla-beled cytidine bisphosphate; HA, hemagglutinin.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 4, pp. 2535–2548, January 23, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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well as the gene silencing activities of the siRNAs (23).Together, these data suggested that Dicer participates in thetranslocation of the cleavage product to RISC and that bindingdirectionality between the enzyme and the product plays a rolein the selective loading of the siRNA strands into RISC.The substrate specificity of humanDicer has been character-

ized. The enzyme cleaves long double-strand RNA substratesfromeither terminus, producing cleavage products that are typ-ically 21–25 base pairs long (10). Substrates containing 3�-over-hangs exhibited faster cleavage rates compared with substrateswith blunt termini, although both substrates exhibited similarbinding affinities for the enzyme (24). The sequence composi-tion of the 3�-overhangs as well as the length of the overhangsalso affected the rate of cleavage, with no cleavage observed foroverhangs longer than 3 ribonucleotides (24).It is surprising, given that the enzyme is believed to partici-

pate in the translocation of the products to RISC thatmuch lessis known about the interaction between human Dicer andsiRNA products it produces. Provost et al. (25) showed by elec-trophoretic mobility shift assay Dicer did not bind siRNA, sug-gesting that the enzyme may not be directly involved in thetranslocation of the products of Dicer cleavage (siRNAs) intoRISC. Thus, there is a need to resolve the issue of whetherDicerindeed translocates siRNA to RISC and how this is accom-plished. To better understand the interaction between humanDicer and its siRNA cleavage products as well as the propertiesimportant for the translocation of the siRNA from Dicer toRISC, we determined the binding affinities of human Dicer forsubstrate and various native and chemically modified siRNAsand short single-strand RNAs (ssRNA). Based on the observedbinding interactions between the enzyme and the siRNAs andssRNAs, substrates containing short siRNA-like double-strandregions and extended 3� or 5� single-strand RNA regions wereprepared, and the Dicer cleavage activities were determined.Finally, we compared the gene silencing activities of variousmodified and unmodified siRNAs in cultured cells with thebinding affinities of human Dicer for the siRNAs.

EXPERIMENTAL PROCEDURES

Preparation of Oligonucleotides and 32P-Labeled Substrate—Synthetic oligoribonucleotidesweremanufactured byDharma-con Research, Inc. siRNA duplexes were formed accordingto the manufacturer’s instructions. The double (siRNA) andsingle strand (ssRNA) structures of the oligoribonucleotideswere confirmed by native polyacrylamide gel electrophoresis.The alternating 2�-fluoro-, 2�-methoxy-modified oligonucleo-tides were prepared as previously described (26). The final con-centration of the duplex was 20 �M in 100 mM potassium ace-tate, 2 mM magnesium acetate, 30 mM HEPES-KOH, pH 7.4.The 27-base pair RNA substrate was 5�-end-labeled with[�-32P]ATP, T4 polynucleotide kinase, and standard proce-dures (27). The labeled substrate was purified on a 12% dena-turing polyacrylamide gel. The specific activity of the labeledoligonucleotide was �3000–8000 cpm/fmol.Preparation of Recombinant Human Dicer—The human

Dicer cDNA clone was purchased fromOriGene Technologies,Inc. (Rockville, MD). The GST and His tags were engineeredinto theNandC termini of the cDNAof the pcDNA3.1/V5-HIS

plasmid (OriGene Technologies). The GST-Dicer-His cDNAwas then subcloned into the pENTR 2B plasmid (Invitrogen).The plasmid was recombined with the BaculoDirect LinearDNA to create the BaculoVirus expressing GST- and His-tagged human Dicer in sf9 cells. The virus was amplified andtransfected into sf9 cells. The cells were harvested 3 days fol-lowing transfection, and lysate was subjected to two affinitypurifications. First, His tag affinity purification with the Taloncolumn (Clontech) and elution of the enzymewith 80mM imid-azole and 80mMEDTAwere performed. Second, the eluantwasGST affinity-purified with the GST column according to themanufacturer’s instructions (Amersham Biosciences). Thepurity of the Dicer protein was more then 95%.Multiple-turnoverKinetics—The27-base pair RNAsubstrate

ranging in concentration from 100 nM to 10 �M was digestedwith 200ng of humanDicer in cleavage buffer (20mMTris-HCl,pH 8.0, 150 mM NaCl, 2.5 mM MgCl2) at 37 °C. At times of2–120 min, a 10-�l aliquot of the cleavage reaction wasquenched with 5 �l of stop solution (8 M urea and 120 mMEDTA). Cleavage reactions were analyzed by denaturing poly-acrylamide gel electrophoresis and quantitated on an Amer-sham Biosciences PhosphorImager. The concentration of theconverted product was plotted as a function of time. The initialcleavage ratewas obtained from the slope (mol of RNAcleaved/min) of the best fit line for the linear portion of the plot, whichcomprises in general �10% of the total reaction and data fromat least five time points. Alternatively, cleavage products wereanalyzed by ion pair HPLC electrospray mass spectrometry.Specifically, samples were phenol/chloroform-extracted andinjected directly on a Waters XBridge column (Waters, Mil-ford, MA). Separation was accomplished using an Agilent 1100HPLC-mass spectrometry system (Agilent, Wilmington, DE).The column was maintained at 55 °C, and flow rate on the col-umn was 0.25 ml/min. The column was equilibrated with 35%acetonitrile in 5 mM tributylammonium acetate, pH 7.0. A gra-dient from 35 to 80% acetonitrile over 15 min was used to elutethe RNA duplex and dicer processing products, and the singlestrands were detected in the mass spectrometer. Mass spectrawere obtained using a spray voltage of 4 kV, a sheath gas flow of35 p.s.i.g., a drying gas flow rate of 12 liters/min at 325 °C, and acapillary voltage of �125 V. under these conditions, oligonu-cleotides ranging in length from 15 to 27 nucleotides aredetected in the �4 and �5 charge state. Chromatograms andmass spectra were analyzed using Agilent Chemstation soft-ware. Each peak was manually averaged for its m/z value, andthe results were compared with a table containing the calcu-latedm/z values of expected Dicer products.Determination of Dissociation Constants (Kd)—Binding

affinities were determined by inhibition analysis. Here, the ini-tial cleavages rates were determined for the 27-base pair RNAsubstrate at a variety of concentrations in both the presence andabsence of a competing noncleavable substrate analog (i.e.siRNA, ssRNA, and dsDNA), as described for multiple-turn-over kinetics. The dsRNA substrate was prepared as describedabove except in 50 �l of cleavage buffer with final concentra-tions ranging from 200 to 800 nM. The competing noncleavablesubstrate analog was prepared in 50 �l of hybridizationbuffer ranging in concentration from 1 to 10 �M. The reac-

Human Dicer Binds Short Single-strand and Double-strand RNA

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tions were digested with human Dicer, quenched, analyzed,and quantitated as described for multiple-turnover kinetics.The equation, Ki � [inhibitor]/((slopeinhibitor/slopesubstrate) � 1)was used to calculate the inhibition constant (Ki). Ki equals thedissociation constant (Kd) when the competitor is a noncleav-able substrate.

siRNA Treatment—HeLa cellswere cultured in DMEM supple-mented with 10% fetal calf serum,streptomycin (0.1 �g/ml), and pen-icillin (100 units/ml). Treatment ofcells with siRNA was performedusing Opti-MEM medium (Invitro-gen) containing 2–5 �g/ml Lipo-fectamine 2000 (Invitrogen) for 3–5h at 37 °C, as described previously.For the generation of IC50 curves,cells were seeded in 96-well plates at4000–6000 cells/well and thentreated at doses ranging from 2 pMto 60 nM in half-log serial dilutions.Treated cells were incubated over-night. The next day, total RNA waspurified from 96-well plates usingan RNeasy 3000 BioRobot (Qiagen,Valencia, CA). Reduction of targetmRNA expression was determinedby quantitative RT-PCR. mRNAlevels were normalized to total RNAfor each sample as measured byRibogreen (Invitrogen). IC50 curvesand values were generated usingPrism 4 software (GraphPad).Immunoprecipitation of Overex-

pressed Ago2 for the RISC ActivityAssay—The cDNA of human Ago2with an N-terminal HA epitope wassubcloned into the mammalianexpression vector phCMV-2. HeLacells (CCL-2) were treated with theAgo2 expression plasmid usingEffectene transfection reagent. 24 hlater, cells were treated with 75 nMsynthetic oligoribonucleotides orsiRNAs using Lipofectamine 2000reagent and incubated for an addi-tional 18 h. Cells were harvestedwith trypsin and washed twice with1 ml of cold phosphate-bufferedsaline. The cell pellet was resus-pended in 500 �l of lysis buffer (150mM NaCl, 0.5% Nonidet P-40, 2 mMMgCl2, 2 mM CaCl2, 20 mM Tris atpH 7.5, protease inhibitor, and 1mMdithiothreitol) and passed throughan insulin syringe. Supernatantswere clarified with a 10,000 � gclearing spin for 10min, and protein

concentrations were determined. 1.8 mg of total protein wasincubated with 15 �l of HA II beads (Covance) for 2 h, washedthree times with lysis buffer, and equilibrated in cleavage buffer(10 mM Tris at pH 7.5, 100 mM KCl, 2 mM MgCl2, proteaseinhibitor, 0.5mMdithiothreitol). 0.1 nM 32P-labeled target RNAwas added, and cleavage reactions quenched at the specified

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FIGURE 1. Ion pair HPLC electrospray mass spectrometry analysis of 27-base pair substrate digested withhuman Dicer. A, mass spectra of the 27-base pair substrate incubated with (top and center) and without(bottom) human Dicer. The charge states for the products are shown above the corresponding peak.B, sequences of the detected products are provided along with the strand lengths (n), the calculated molecularweights, and observed molecular weights based on the observed charge states from A. C, sequence of sense(top) and antisense (bottom) strands of the 27-base pair substrate are shown, respectively, 5�3 3� and 3�3 5�.The arrows indicate the positions of the Dicer cleavage sites corresponding to the most abundant cleavageproducts detected by liquid chromatography-mass spectrometry.




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times in gel loading buffer (Ambion). Cleavage reactions wereanalyzed by denaturing polyacrylamide gel electrophoresis andquantitated with an Amersham Biosciences PhosphorImager.

RESULTS

siRNA and ssRNA Are Competitive Inhibitors of DicerSubstrate—Kd values for substrate, the cleavage products ofDicer (siRNA), and short ssRNAwere determined by inhibitionanalysis. We chose inhibition analysis for several reasons. First,this method allows the measurement of a wide range of disso-ciation constants, including weaker binding affinities (i.e. thosein the high nanomolar to micromolar range) (28). Second, thismethod provides information about the nature of the enzyme/ligand interaction. Specifically, the type of inhibition, (e.g. com-petitive, uncompetitive, noncompetitive) gives an indication ofwhether the inhibitor is binding to the reactive site on the

enzyme, modifying the substrate, orcausing an allosteric effect on theenzyme. Lineweaver-Burk andAugustinsson analysis of the datawere used to determine the inhibi-tory constant (Ki), and the Ki isequivalent to the Kd of the enzymewhen a noncleavable inhibitor isused (28).The enzyme cleaved the 27-base

pair substrate, generating 21–25-basepair products (Figs. 1 and 2A). Todetermine the binding affinity ofhuman Dicer for substrate, a27-base pair noncleavable substratewas prepared with 2�-methoxyribo-nucleotide substitutions at thecleavage sites (Fig. 2A). No humanDicer cleavage activity was observedfor the noncleavable 27-base pairduplex containing 2�-methoxy sub-stitutions, the 19-base pair siRNA(si92), or the 19-nucleotide single-strand RNA (s92) (Fig. 2A). Lin-eweaver-Burk analysis of the initialcleavage rates for the 27-base pairRNA substrate in the presence andabsence of the si92 siRNA or s92single-strand RNA demonstratedthat si92 and s92 were competitiveinhibitors of the 27-base pair sub-strate (Fig. 2, B and C). The non-cleavable 27-base pair duplex con-taining 2�-methoxy ribonucleotidesubstitutions at the cleavage sitesalso functioned as competitiveinhibitor of the 27-base pair sub-strate (data not shown). The Kdvalues for the noncleavable27-base pair duplex, si92, and s92were 47, 451, and 154 nM, respec-tively (Fig. 2A).

Human Dicer Binds siRNA Products and Short Single-strandRNAs—Previous studies have shown that the thermodynamicstabilities of the terminal base pairs of the siRNA duplex as wellas 3�-overhangs appear to influence the binding directionalityof the siRNA to the RLC (22). To evaluate the effects of ther-modynamic stabilities of the terminal base pairs on the interac-tion with the enzyme, we designed 19-base pair siRNAs con-taining terminal base pairs predicted to exhibit similar (si92)and asymmetrical (si97) thermodynamic stabilities and siRNAswith various types of overhangs (Table 1).The dissociation constants for the 19-base pair si92 and si97

duplexes were �7–12-fold weaker compared with the Kd forthe 27-base pair noncleavable substrate (Table 1A and Fig. 2A).Dicer exhibited a slightly tighter binding affinity for the si97duplex with G:C and A:U base pairs at the termini, comparedwith the si92 duplex with A:U terminal base pairs (Table 1A).

FIGURE 2. siRNA and single-strand RNA are competitive inhibitors of human Dicer substrate. A, denatur-ing polyacrylamide gel analysis of substrates incubated without human Dicer (lanes 1, 3, 5, and 7) or withhuman Dicer (lanes 2, 4, 6, and 8). The arrows indicate the bands corresponding to the Dicer cleavage products.Digestion reactions were prepared as described under “Experimental Procedures.” Reactions were incubatedat 37 °C for 90 min. The sequences of the various substrates are shown beside the polyacrylamide gel. Theunderlined sequences represent 2�-methoxy substitutions. The arrows indicate positions of Dicer cleavage.Dissociation constants (Kd) were determined by competitive inhibition. The positions of the 32P label (p) areshown in boldface type. Dicer digestion of 27-base pair substrate (lanes 1 and 2), 27-base pair noncleavablesubstrate (lanes 3 and 4), 19-base pair siRNA (si92) (lanes 5 and 6), and 19-nucleotide single-strand RNA (s92)(lanes 7 and 8). B and C, Lineweaver-Burk analysis of human Dicer cleavage activities of the 27-nucleotide RNAduplex alone (triangle) and in the presence of 300 nM s92 single strand RNA (box) or 900 nM si92 siRNA (circle).Initial cleavage rates (V0) were determined as described under “Experimental Procedures.”




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The 5�-phosphate appeared to have no effect on Dicer binding,since comparable dissociation constants were also observed forthe si92 duplexes containing 5�-phosphates (si92/s5�-PO, si92/as5�-PO, and si92/s:as5�-PO) and the si92 siRNA (Table 1A).Similarly, comparable binding affinities were observed for thesi97 duplex containing a 5�-phosphate (si97/as5�-PO) and thesi97 siRNA (Table 1A). Finally, human Dicer exhibited a signif-icantly weaker binding affinity for the double-strand deoxyri-bonucleotide duplex (si92/DNA) compared with si92.

To explore the effects of the dinucleotide 3�-overhang onDicer binding, a series of duplexes was prepared in whichpurine andpyrimidine ribonucleotide anddeoxyribonucleotideoverhangs were positioned at either the 3� or 5� terminus of thesense and antisense strands of the si92 and si97 duplexes (Table1B). The duplexes containing 5�-dinucleotide overhangs exhib-ited Kd values comparable with the length-matched si92duplex, suggesting that the 5�-overhangs have no effect on thebinding affinity of human Dicer for the siRNA (Table 1B). The

TABLE 1Dissociation constants for human Dicer binding to siRNA constructsKd values were determined by competitive inhibition as described under “Experimental Procedures.” The reported Kd and S.E. values are the mean values from threeexperiments. The �Kd is the ratio of the Kd for the siRNA construct divided by the Kd for the noncleavable substrate determined to be 47 nM. siRNA constructs containedribonucleotides (black), deoxyribonucleotides (red), and 5�-phosphate (PO) in the sense (top) and antisense (bottom) strands. The sequences for the sense and antisensestrands are shown, respectively, 5�33� and 3�35�. A, si92 and si97 length-matched siRNAs constructs. B, si92 and si97 siRNA constructs containing 5�- and 3�-purine orpyrimidine diribonucleotide substitutions (underlined). 3�-Dideoxyribonucleotide substitutions are shown in red.




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position of the 3�-overhang (e.g. 3� terminus of the sense strandverses the antisense strand) of the siRNA appeared to have agreater influence on human Dicer binding compared with thenucleotide composition of the 3�-overhang. Specifically, humanDicer exhibited a 2–7-fold tighter binding affinity for the si92duplexes containing the 3�-diribonucleotide overhang in theantisense strand compared with the si92 duplexes with the3�-overhang in the sense strand (Table 1B). In contrast, varyingthe dinucleotide composition in the 3�-overhang of the anti-sense strand resulted in only a 2-fold difference in binding affin-ity. Similarly, a maximum 2-fold difference in binding affinitywas observed for the various nucleotide combinations at the3�-overhang of the sense strand. (Table 1B). Finally, the influ-ence of the 3�-overhang on human Dicer binding appears to beindependent of duplex sequence. For example, a tighter bindingaffinity was also observed for the si97 duplex containing thethymidine deoxyribonucleotide overhang at the 3� terminus ofthe antisense strand (si97/as3�-dTdT) compared with theduplex overhang at the 3�-terminus of the sense strand (si97/s3�-dTdT) (Table 1B). Taken together, these data suggest thatalthough the thermodynamic stability of the terminal base pairsplays a role, the dinucleotide 3�-overhangs exhibit a greatereffect on human Dicer binding.Table 2 shows the binding affinities of human Dicer for sin-

gle-strand RNAs. The binding affinities for the various single-strand oligonucleotides differed significantly compared withthe siRNA duplexes. Specifically, human Dicer exhibited an�14-fold tighter binding affinity for the sense strand (s92) com-pared with the antisense strand (as92) of the si92 duplex (Table2A). The tightest binding affinitieswere observed for the single-strand RNAs that contained purine dinucleotides at the 3� ter-minus compared with single-strand RNAs with 3�-terminal py-rimidine dinucleotides. Remarkably, the binding affinity for s92was only 3-fold weaker than the binding affinity for the non-cleavable 27-base pair substrate (Table 2A). A similar trendwasobserved for the single-strandRNAs of the si97 duplex inwhichDicer bound the sense strand tighter than the antisense strand,although a smaller 2�-fold difference in binding affinity wasobserved (Table 2B). The 2�-fluoro/2�-methoxy substitutionsreversed the rank order for binding. Specifically, Dicer exhib-ited a 2-fold tighter binding affinity for the modified antisensestrands (as92/2�-F/OMe and as97/2�-F/OMe) compared withthe modified sense stands (s92/2�-F/OMe and s97/2�-F/OMe)(Table 2, A and B). Finally, significantly weaker binding affini-ties were observed for the single-strand RNAs containing thy-midine deoxynucleotides at the 3� termini of the sense and anti-sense strands (Table 2, A and B).To better understand the observed differences in the human

Dicer binding affinities for single-strand sense and antisenseRNAs, s92 and as92 were designed with additional uridine res-idues positioned at both termini of s92, and adenine residueswere positioned at both termini of as92 (Table 2C). The dinu-cleotide substitution at the 3� terminus of the oligoribonucle-otide significantly affected humanDicer binding affinity. Again,Dicer exhibited the tightest binding affinities for the single-strand RNAs that contained purine dinucleotides comparedwith pyrimidine dinucleotides at the 3� terminus. For example,human Dicer exhibited 8–12-fold tighter binding affinities for

the oligoribonucleotides containing adenine dinucleotides atthe 3� terminus compared with the oligoribonucleotides con-taining 3�-uridine dinucleotides (Table 2C). In contrast, dinu-cleotide substitutions at the 5� terminus appeared to have noeffect on Dicer binding, since comparable binding affinitieswere observed for s92 and the s92/5�-UU and as92/5�-AAduplexes (Table 2C). In addition, these data suggest that theinfluence of the dinucleotide substitutions on Dicer bindingaffinity is not due to the increased length of the ssRNA.Human Dicer Cleaves Substrates Containing Extended Sin-

gle-strand Overhangs from the 3� Single-strand Terminus andwithin the 3� and 5� Single-strand Regions—Given the observedstrong binding affinity of human Dicer for siRNAs and single-strand RNAs, we asked whether human Dicer could bind andcleave substrates containing short siRNA-like double-strandregions. We prepared a series of substrates containing double-strand RNA regions with lengths consistent with Dicer sub-strate (e.g. 23–25 base pairs) or Dicer product (e.g. 15–21 basepairs) (Figs. 4 and 5). In addition, substrates containing flanking3�- or 5� single-strand RNA regions ranging from 5 to 13 nucle-

TABLE 2Dissociation constants for human Dicer binding to single-strandRNAsKd and �Kd are reported as described in Table 1. Single-strand RNA sequencescontained ribonucleotides (black), deoxyribonucleotides (red), 5�-phosphate (PO),2�-fluoro (green), and 2�-methoxy (blue) modified nucleotides. The sequences forthe sense (s) and antisense (as) strands are shown 5�33�. A, sense and antisensesequences for the si92 siRNA. B, sense and antisense sequences for the si97 siRNA.C, sense and antisense sequences for the si92 siRNA containing 5�- and 3�-purineand pyrimidine diribonucleotide substitutions (underlined).




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otides in length were tested. Each substrate contained double-strand blunt (B), double-strand overhang (O), and single-strand(S) termini (Figs. 4 and 5). Given that human Dicer cleaves thesubstrate at a fixed distance from either end of the duplex, weused the position of Dicer cleavage to determine the bindingdirectionality of the enzyme on the substrates (24). To evaluatethe Dicer cleavage activities at both termini, the substrates wereprepared with the 32P label at either the 5� terminus of the sensestrand, 3� terminus of the sense strand, or 5� terminus of the anti-sense strand. Finally, the Dicer cleavage of these substrates wascompared with cleavage of 25-base pair length-matched sub-strates of the same sequence (Fig. 3).Human Dicer cleavage of the 25-base pair length-matched

substrates resulted in the predicted Dicer cleavage productsranging from 19 to 23 base pairs (Fig. 3) (24). The cleavagesites at positions 3–5 and 21–23 indicated that the enzymecleaved the substrates from both blunt termini. The extent ofthe cleavage activity appeared to be similar for both3�-sense/5�-antisense and 5�-sense/3�-antisense terminicleavage products (Fig. 3).The same Dicer cleavage sites at positions 3–5 and 21–25

were observed for the substrates containing 23- and 25-basepair double-strand regions and 3� single-strand overhangs inthe sense strand (23/3�-7a and 25/3�-5a and -b) (Figs. 3 and 4,Aand B). The positions of these cleavage sites were consistent

with Dicer entering the substratesfrom both the blunt end (B) andoverhang termini (O) of the double-strand regions (Fig. 4, A and B).Interestingly, new cleavage siteswere observed at positions 7 and 10in the sense strand of the 23/3�-7aand 25/3�-5a substrates and 18 and19 in the antisense strand of the25/3�-5b substrate that were notobserved in the 25-base pair length-matched substrate (Figs. 3 and 4, Aand B). The new cleavage productsappear to be too short to be gener-ated by Dicer entering the sub-strates from either the blunt end oroverhang termini of the duplex.Instead, the new cleavage sites are20–23 nucleotides from the 3� sin-gle-strand RNA terminus of thesubstrate, suggesting that the cleav-age sites were generated from the 3�single-strand RNA terminus (Scleavage products) (Fig. 4, A and B).Similar cleavage sites at positions5–10 were observed for the sub-strates containing double-strandregions 21 base pairs or shorter (21/3�-9a, 19/3�-11a, 17/3�-13a, 17/3�-13b, and 15/3�-15) and, given theshorter length of the double-strandregions of these substrates, strong-ly suggests that these cleavage sites

are generated from the 3� single-strand termini (S) and notthe blunt end (B) or overhang (O) termini (Fig. 4, A and B).Finally, thymidine deoxyribonucleotide overhangs at the 3�terminus of the antisense strand appeared to have no effecton the positions of Dicer cleavage (17/3�-13b and 17/3�-15)(Fig. 4, A and B).To evaluate the Dicer cleavage activities closest to the 3� sin-

gle-strand RNA overhang, the substrates were labeled at the 3�terminus of the sense strand with 32P-labeled cytidine bisphos-phate (3�-32PCp) (Fig. 4, C andD). Once again, cleavage sites atpositions 22–25 were observed for the 3�-32PCp labeled sub-strates (25/3�-5c and 23/3�-7b) (Fig. 3). The 3�-32PCp-labeledsubstrates containing 17–21-base pair double-strand regions(21/3�-9b, 19/3�-11b, 17/3�-13c, and 17/3�-13d) exhibited a sin-gle cleavage site at position 22 (Fig. 4,C andD). Remarkably, thecleavage site for these substrates was positioned in the single-strand RNA overhang region. In contrast to the 5�-32P-labeledsubstrates, no cleavage sites at positions 7 and 10were observedfor the 3�-32PCp-labeled substrates (Fig. 4). The lack of Dicercleavage at these positions suggested that the 3�-32PCp wasinterfering with the enzyme activity at these sites. To test thishypothesis, unlabeled cytidine bisphosphate (pCp) was ligatedto the 3� terminus of the sense strands of the 5�-32P-labeled25/3�-5 and 19/3�-11 substrates (25/3�-5d and 19/3�-11c) (Fig.4, C and D). Again, no cleavage sites at positions 7 and 10 were

FIGURE 3. Human Dicer cleavage pattern for length-matched 25-base pair duplexes. Shown is denaturingpolyacrylamide gel analysis of substrates incubated with (�) and without (�) human Dicer. Reactions wereincubated at 37 °C for 90 min. Substrate structures are shown below the gels with the nucleotide positions onthe sense strand (top sequence) numbered from 5� to 3� and antisense strand (bottom sequence) numberedfrom 3� to 5�. Substrates were prepared with either a 32P-labeled sense strand (gels 1 and 3) or a 32P-labeledantisense strand (gels 2 and 4). The positions of the 32P label (p) are shown in boldface type. The arrows indicatethe positions of the Dicer cleavage sites for each substrate. Each 32P-labeled strand was digested with RNase T1(T1) to determine the positions of the Dicer cleavage sites.




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observed for 5�-32P-labeled 25/3�-5d substrate with 3�-pCp(Fig. 4, C and D). Similarly, no Dicer cleavage sites wereobserved at positions 6, 7, and 10 of the 5�-32P-labeled 19/3�-11c substrate with 3�-pCp. The addition of a cytidine residue atthe 3� terminus of the sense strand of the 25/3�-5a and 19/3�-11a substrates did not inhibit Dicer cleavage at positions 7–10(data not shown). Taken together, these data suggest that the3�-phosphate in the sense strand of the substrate is interfering

with the interaction between theenzyme and 3� single-strand RNAregion of the substrate.Fig. 5 shows the Dicer cleavage

activity for the substrates contain-ing double-strand RNA regionswith flanking 5� single-strand RNAregions. Similar cleavage patternswere observed for the 25-base pairlength matched substrate and thesubstrates containing a 25-base pairdouble-strand region with a flank-ing 5� single-strand region (25/5�-5a, -b, and -c) (Figs. 3 and 5). Thecleavage sites were consistent withthe enzyme entering the substratefrom the blunt end (B) and overhangterminus (O) of the substrates (Fig.4). Importantly, no Dicer cleavagesites generated from the flanking 5�single-strand RNA regions of the25/5�-5 substrates were observed(Fig. 5). A single cleavage site atposition 7 was observed for the17–21-base pair substrates (17/5�-13, 19/5�-11, and 21/5�-9) (Fig. 5).Once again, the cleavage sites werepositioned within the adjacent sin-gle-strand RNA region (Fig. 5).3�-Overhangs in siRNA Influence

RISC Loading—Dicer exhibited atighter binding affinity for the si92siRNA containing the 3�-overhangin the antisense strand (si92/as3�-dTdT) compared with the duplexwith the 3�-overhang in the sensestrand (si92/s3�-dTdT) (Table 1B).To determine whether the bindingaffinities of Dicer for these siRNAsinfluence the selective loading of thesiRNA strands into RISC, the si92/as3�-dTdT, si92/s3�-dTdT, andlength-matched si92 siRNAs weretransfected into HeLa cells express-ing HA-tagged human Argonaute2enzyme (HA-Ago2). HA-Ago2 wasthen immunoprecipitated from thetransfected cells and incubated witha 40-nucleotide 32P-labeled senseRNA, and the cleavage activity of

HA-Ago2 was used to estimate the relative amounts of thesiRNA strands loaded into Ago2 (Fig. 6). Given that the 32P-labeled sense RNA is complementary to the antisense strand ofthe siRNA, if the antisense strand of the siRNA is favorablyloaded into Ago2 compared with the sense strand, the Ago2cleavage activity should be enhanced. Alternatively, if the sensestrand of the siRNA is favorably loaded into Ago2, the cleavageactivity should be reduced.

FIGURE 4. Human Dicer cleavage pattern for substrates containing 3�-sense strand overhangs. A, dena-turing polyacrylamide gel analysis of 3�-overhang substrates with 32P label at the 5� terminus of the sensestrand, incubated with (�) and without (�) human Dicer. Dicer digestion reactions were performed asdescribed under “Experimental Procedures.” Shown is T1 digestion (T1) of 30-nucleotide sense strand (lane 1).The schematic below the gel shows the positions of the predicted Dicer cleavage products generated from theblunt terminus (B), overhang terminus (O), or 3� single-strand region (S; shown in red) of the substrate. B, thestructures of the 3�-overhang substrates with 32P label at the 5� terminus of the sense strand along withthe respective lane designations for the gel. Substrate structures are shown with the nucleotide positions onthe sense strand (top sequence) numbered from 5� to 3� and antisense strand (bottom sequence) numberedfrom 3� to 5�. The positions of the 32P label (p) are shown in red. The black arrows indicate the positions of theDicer cleavage sites for blunt terminus and overhang terminus products. The red arrows indicate the positionsof the Dicer cleavage sites for single-strand region products. C, denaturing polyacrylamide gel analysis of3�-overhang substrates with 3�-pCp substitutions, incubated with (�) and without (�) human Dicer. Shown isT1 digestion (T1) of the 30-nucleotide sense strand with 3�-32pCp label (lane 1) or 5�-32P label (lane 9). Theschematic below the gel is as described in A. D, structures of the 3�-overhang substrates with 3�-pCp substitu-tions along with the respective lane designations for the gel. Substrate structures are shown as described in B.The positions of the 32P (p) are shown in red. The black arrows indicate the positions of the Dicer cleavage sitesfor blunt terminus and overhang terminus products.




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The immunoprecipitated Ago2 enzyme from the cellstreated with the various siRNAs cleaved the target RNA in anAgo2-dependent manner (i.e. a single cleavage site betweenthe 10th and 11th nucleotide from the 5� terminus of theantisense strand was observed in the target RNA) (Fig. 6, Aand B). The cleavage activity observed for si92/as3�-dTdT,which exhibited the tightest binding affinity for humanDicer, was more than 2-fold greater compared with thecleavage activities observed for si92 and si92/s3�-dTdT (Fig.6C). In other words, 100% more antisense strand than sensestrand appeared to be loaded into the enzyme (Fig. 6C). Inaddition, these data suggest that the 3�-thymidine deoxyri-

bonucleotide overhang biasesAgo2 loading in favor of the strandwith the thymidine deoxyribonu-cleotide overhang.Chemical Modification of the

siRNA Enhanced Dicer Binding andGene Silencing Activity in CellCulture—Dicer binding affinitiesand gene silencing activities of theunmodified and chemically modi-fied si92 and si97 siRNAs are shownin Table 3. The gene silencing activ-ities of the siRNAs were determinedby measuring the reduction inPTEN target mRNA from HeLacells transfected with various con-centrations of the siRNAs. The 50%inhibitory concentration (IC50) forthe various siRNAs were calculatedfrom the dose-response curves.The dissociation constants for

the si92 duplexes containing alter-nating 2�-fluoro-, 2�-methoxy-modified ribonucleotides (si92/2�-F/OMe and si92/2�-F/OMe:as5�-PO) were �2-fold tighter thantheKd observed for unmodified si92(Table 3A). The modified duplexesalso showed a similar improvementin potency with an �2–3-fold lowerIC50 observed for si92/2�-F/OMeand si92/2�-F/OMe:as5�-PO com-pared with unmodified si92 andsi92/as5�-PO (Table 3A). A similartrend was observed for the duplexescontaining 3�-overhangs. The si92/as3�-dTdT duplex exhibited atighter binding affinity and greaterpotency compared with the si92/s3�-dTdT and length-matched si92duplexes (Table 3A).The modified si97 duplexes also

exhibited tighter binding affinitiesand greater gene silencing activi-ties (Table 3B). In this case, themodified si97 duplexes exhibited a

significantly greater enhancement in the gene silencingactivities compared with the observed enhancement in bind-ing affinities (Table 3B). For example, the modified si97duplexes exhibited 5–7-fold tighter binding affinities and20–30-fold lower IC50 values compared with the unmodifiedsi97 duplexes (Table 3B). Taken together, these data suggestthat the effects of modified nucleotide substitutions on bothDicer binding and siRNA potency may be influenced by thesequence of the duplex and that the binding affinity of Dicerfor siRNAs affects potency but that the correlation is notprecise, suggesting that other factors may be involved in thegreater potency of the modified si97 siRNAs.

FIGURE 5. Human Dicer cleavage pattern for substrates containing 5�-sense strand overhangs. A, dena-turing polyacrylamide gel analysis of 5�-overhang substrates incubated with (�) and without (�) human Dicer.Digestion reactions were performed as described under “Experimental Procedures.” Shown is T1 digestion (T1)of 30-nucleotide sense strand with 5�-32P label (lane 1), 25-nucleotide antisense strand with 5�-32P label (lane 8),and 30-nucleotide sense strand with 3�-32pCp label (lane 11). The schematic below the gel is as described in thelegend to Fig. 4A. B, structures of the 5�-overhang substrates along with the respective lane designations forthe gel. Substrate structures are shown as described in the legend to Fig. 4B. The positions of the 32P (p) areshown in red. The black arrows indicate the positions of the Dicer cleavage sites for blunt terminus (B) andoverhang terminus (O) products.




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DISCUSSION

HumanDicer Avidly Binds siRNAProduct and Short Single-strandRNAs—We determined the bindingaffinity of Dicer for two differentsiRNA sequences, ranging in lengthfrom19 to 21 base pairs and consist-ing of length-matched siRNAs,siRNAs with 3� and 5�-dinucleotideoverhangs, and chemicallymodifiedsiRNAs. The siRNAs and ssRNAsfunctioned as competitive inhibitorsof substrate, suggesting that thesenucleic acid structures bind to theactive site of the enzyme (Fig. 2). Therange of binding affinities observedfor the various siRNAs included Kdvalues that were comparable to sub-strate to 20-fold weaker than sub-strate (Tables 1 and 3). The ssRNAsexhibited binding affinities rangingfrom 3- to 50-fold lower than thebinding affinity for substrate (Table2).Our results differ fromapreviousreport that showed that Dicer didnot bind siRNAs using an electro-phoreticmobility shift assay (25). Atleast two factors may explain thedifferences between our study and

that of Provost et al. (25). Compared with the electrophoreticmobility shift assay, the competitive inhibition assay used herehas been shown to be useful in measuring a wider range ofdissociation constants, particularly weaker binding affinities(e.g. Kd values in the high nanomolar to lowmicromolar range)(28). A second factor is that in this study, we addressed thisquestion more intensively with multiple siRNAs. Importantly,the Kd of 47 nM observed for the noncleavable 27-base pairsubstrate by competitive inhibition is consistent the with the50–60 nMKd values observed for longer substrates determinedusing either nitrocellulose filter binding or an electrophoreticmobility shift assay (24, 25).The predominant factor influencing the binding affinity of

human Dicer for siRNA was the position of the 3�-overhang inthe siRNA (Table 1). The composition of the 3�-overhangappeared to play a secondary role with respect toDicer binding.For example, the Dicer binding affinities for the si92 siRNAswith 3�-overhangs varied as much as 7-fold, exhibiting a rankorder for Dicer binding to the si92 siRNA as follows: 3�-over-hang in the antisense strand length-matched duplex �3�-overhang in both the sense and antisense strand 3�-over-hang in the sense strand (Table 1). Within the siRNA groupscontaining the 3�-overhangs in either the sense or antisensestrands, the nucleotide composition within the overhangaccounted for a smaller, 2-fold, variance in binding affinity. Asimilar trendwas observed for the si97 siRNA sequences (Table1). The influence of the 3�-overhang on Dicer binding is prob-ably due to the PAZ domain of Dicer, which has been shown to

FIGURE 6. 3�-Overhangs in the siRNA bias RISC loading. HA-tagged human Ago2 was immunoprecipitatedfrom HeLa cells transfected with the siRNA constructs and incubated with the 40-nucleotide 32P-labeled targetfor 1 h. A, sequence of 19-nucleotide antisense strand and 40-nucleotide target. The arrow indicates theposition of human Ago2 cleavage in the target RNA. B, denaturing PAGE analysis of human Ago2 cleavage ofthe 40-nucleotide target for cells treated with 1 �M siRNA constructs. RNase T1 digestion (T1) of the 40-nucle-otide target, which cleaves at guanidine residues. Lane 1, length-matched siRNA duplex; lane 2, siRNA contain-ing 3�-dinucleotide overhang in the sense strand; lane 3, siRNA containing 3�-dinucleotide overhang in theantisense strand. C, human Ago2 cleavage activity quantitated from denaturing PAGE. The percentage cleavedcorresponds to the counts for the cleavage product/total counts for each lane.

TABLE 3Comparison of Dicer binding affinity with potency in cultured cellsfor siRNA contructsKd and 50% inhibition constants (IC50) were determined as described under “Exper-imental Procedures.” Kd and �Kd are reported as described in Table 1. siRNA con-structs contained ribonucleotides (black), 5�-phosphate (PO), 2�-fluoro (green), and2�-methoxy (blue) modified nucleotides. A, si92 siRNA constructs. B, si97 siRNAconstructs.




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interact with 3�-overhang structures and direct the positioningof the enzyme on the substrate (14, 24). The tighter bindingaffinities observed for the siRNAs containing 3�-overhangs inthe antisense strand compared with the sense strand suggestthat the preferred binding directionality for these duplexes iswith the PAZ domain of the enzyme positioned at the 3� termi-nus of the antisense strand (Fig. 7A). In the case of the siRNAscontaining 3�-overhangs at both the sense and antisensestrands and the length-matched siRNAs for which no preferen-tial binding directionality is predicted, the observed bindingaffinities are probably the sum of the interactions with bothtermini (Fig. 7A). In fact, these siRNAs exhibited Kd values thatwere intermediate between those observed for siRNAs with3�-overhangs in either the sense or antisense strands (Table 1).Because differences in binding directionality result in Dicerbinding to different sequences within the siRNA, our data sug-gest that the interaction of Dicer with the siRNA is sensitive tobase composition. In fact, the basic amino acid residues of thesubstrate-binding surface of Dicer are predicted to form exten-sive electrostatic interactions with the phosphates of the sub-strate, and differences in the helical conformation would bepredicted to have an affect on these interactions (14). Consist-ent with this observation, Dicer bound the B-formDNAduplexmore weakly than the A-form RNA duplex (Table 1A). Inter-estingly, we did not see a difference in Dicer binding affinitiesfor the siRNAs containing 3�-DNA overhangs compared withsiRNAs with 3�-RNA overhangs, again suggesting that the basecomposition within the duplex region of the siRNA plays agreater role in Dicer binding than the composition of the3�-overhang (Table 1 (bottom)). Finally, these results differfrom previous reports that showed that siRNAs with 3�-RNAoverhangs were more potent than siRNAs with 3�-DNA over-hangs, suggesting that additional factorsmay be contributing tothe activities of siRNA molecules (24, 29, 30).

Very different results were observed for the ssRNAs. In thiscase, the binding affinity of Dicer for the ssRNA appeared to beinfluenced solely by the 3�-dinucleotide composition. Dicerbound the ssRNAs containing purine dinucleotides at the 3�terminus on average 15-fold tighter comparedwith the ssRNAscontaining 3�-pyrimidine nucleotides. In contrast, the nucleo-tide composition at the 5� terminus or 5�-phosphate substitu-tions had no effect onDicer binding (Table 2). The strong influ-ence of the 3�-terminal nucleotide composition on the bindingaffinity of Dicer for the ssRNAs suggests that the ssRNAs pre-dominantly bind via the PAZ domain of the enzyme (Fig. 7B).The enhanced Dicer binding affinities for ssRNAs with 3�-ter-minal purine dinucleotides is consistent with the co-crystalstructure of the PAZ domain from Argonaute2 bound to RNA,showing that the aromatic amino acids in the hydrophobicbinding pocket of PAZ form stacking interactions with the two3�-terminal nucleotides, although these result differ frommolecular modeling studies, which suggested that uridinedinucleotides would form the most stable complex with thePAZ domain (31–33). The 2�-fluoro and 2�-methoxy substitu-tions in the ssRNAs reversed the rank order for binding, sug-gesting that 2�-sugar substitutions also affect PAZ binding(Table 2). Conversely, deoxyribonucleotide substitutionsappear to have no effect on Dicer binding (Table 2).Dicer Cleavage Activity Is Consistent with the Observed Bind-

ing Affinities for siRNAs and ssRNAs—We show that humanDicer binds siRNA and ssRNA with high affinity, suggestingthat it is possible for Dicer to cleave substrates containingsiRNA or ssRNA structures. We prepared substrates contain-ing short siRNA-like duplex regions with flanking single-strandRNA regions (Figs. 4 and 5). These substrates contained threedifferent termini: double-strand blunt (B), double-strand over-hang (O), and single-strand (S) (Figs. 4, 5, and 8). Finally, weused the position of cleavage to determine the binding direc-tionality of Dicer on the substrate (24).The cleavage pattern observed for the substrates containing

3� single-strand overhangs in the sense strand was consistentwith the enzyme binding to the double-strand blunt (B), dou-ble-strand overhang (O), and single-strand (S) termini of thesubstrate (Figs. 4 and 8B). In the case of the substrates contain-ing a 3� single-strand overhang in the sense strand and double-strand regions shorter than theminimum length of siRNAs, thecleavages occurred within the single-strand overhang regionimmediately outside the double-strand region (Figs. 4,C andD,and 8B). Interestingly, a pCp substitution at the terminus of the3� single-strand overhang ablated the cleavage sites generatedfrom the 3� single-strand terminus (S), suggesting that the3�-pCp substitution interfereswithDicer binding (Figs. 4,C andD, and 8B). The effect of the pCp substitution on Dicer activityis consistentwith the reported structure of PAZ,which predictsthat the 3�-phosphatewould sterically interferewith the nucleicacid binding pocket of PAZ (31).In contrast to the substrates containing a 3� single-strand

overhang in the sense strand, cleavage patterns for the sub-strates containing 5� single-strand overhang regions suggestthat the cleavage sites were generated only from either the dou-ble-strand blunt (B) or overhang (O) termini (Figs. 5 and 8C).No cleavage sites generated from the 5� single-strand termini

FIGURE 7. Models for the interaction of Dicer with siRNA and ssRNA.A, Dicer interacts equally with either termini of length-matched siRNA (top)and siRNAs with sense and antisense 3�-overhangs (middle), 3�-overhang inthe sense strand (bottom left), or antisense strand (bottom right) bias Dicerbinding directionality with the PAZ domain (open circle) positioned at theoverhang. B, PAZ domain of Dicer binds the 3� terminus of the ssRNA, exhib-iting tighter binding affinity for purine dinucleotides (pu) compared with py-rimidine dinucleotides (py) at the 3�-terminus. The enzyme is shown with thePAZ domain (open circle), RNase III domains (solid black circle), and basic plat-form (��).




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(S) were observed (Figs. 5 and 8C). Again, for the substratescontaining double-strand regions shorter than the minimumlength of siRNAs, the cleavages occurred within the single-strand overhang region immediately outside the double-strandregion (Figs. 5 and 8C). The ability of a double-strand-specificRNase to cleave adjacent single-strand RNA regions was alsoreported for Escherichia coli RNase H1 (34). Cleavage occurredexclusively within the 3� single-strand RNA overhang, andcleavage sites were observed up to 3 nucleotides from the end oftheDNA/RNAheteroduplex. In contrast, humanDicer cleavedboth the 3�- and 5�-overhangs at distances up to 7 nucleotidesfrom the duplex (Figs. 3 and 4).Taken together, these data are consistent with the observed

binding specificity of human Dicer. Specifically, Dicer is capa-ble of binding to siRNA regions within a substrate, but giventhat the double-strand region is shorter than the minimumlength for cleavage, the cleavage occurswithin the single-strandoverhang region immediately adjacent the siRNA region. In

addition, Dicer is capable of bindingto 3� single-strand regions but notthe 5� single-strand and cleavingwithin the adjacent double-strandregion. Thus, Dicer is promiscuousin both binding and cleavage behav-ior, permitting it to participate incleavage and RISC loading but alsomaking it vulnerable to nonspecificcleavage activities and to competi-tive inhibition by a wide range ofnucleic acid structures.Dicer Binding Specificity Trans-

lates to siRNA Activity—The ob-served binding affinities of Dicerfor its cleavage products (i.e.siRNAs) and ssRNAs are consist-ent with the enzyme’s role as amember of the RLC family inwhich Dicer transfers its cleavageproducts to RISC. Exactly how thetranslocation of the siRNA fromthe RLC to RISC is accomplishedis unclear, but at some point dur-ing this process, the double-strandsiRNA product of Dicer must beconverted to a single-strand agent(i.e. antisense strand once loadedinto Ago2 must hybridize to thetarget mRNA). It is unclearwhether the conversion of thedouble-strand RNA to the single-strand species occurs before, dur-ing, or after the translocation fromthe RLC to RISC.Our results provide a mechanism

for the reported effects of siRNAstructure on the interaction of thesiRNA with the RLC and the selec-tive loading of the siRNA strands

into RISC (22, 23). Specifically, the results presented here sug-gest that the enzyme is capable of binding and transferringeither the double-strand cleavage product or the unwound sin-gle-strand RNA. For example, the si92 duplex containing the3�-overhang in the antisense strand (si92/as3�-dTdT) of thesiRNA appeared to be preferentially loaded into Ago2, sincegreater Ago2 cleavage of the target sense RNA was observedcompared with the siRNA containing the 3�-overhang in thesense strand (si92/s3�-dTdT) (Fig. 6). Dicer also exhibited atighter binding affinity for the si92/as3�-dTdT siRNA com-pared with the si92/s3�-dTdT siRNA (Table 1). In addition, thesi92/as3�-dTdT siRNA was a more potent inhibitor of geneexpression compared with the si92/s3�-dTdT siRNA (Table 3(top)). These results are consistent with previous data showingthat siRNA duplexes with 3�-overhangs in the antisense strandwere generally more potent than siRNAs with the 3�-overhangin the sense strand (24). Taken together, these results suggestthat the enhanced potency of the si92/as3�-dTdT siRNA

FIGURE 8. Models for the interaction of Dicer with substrates containing 3� or 5�-overhangs. A, Dicercleaves 25-base pair length-matched substrates equally from either blunt terminus. B, interaction of Dicer withsubstrates containing 3� single-strand RNA overhangs. Dicer cleaves the 23- and 25-base pair substrates con-taining 3�-overhangs from the blunt terminus (B), overhang terminus (O), or single-strand RNA terminus (S).Dicer cleaves the 15–21-base pair substrates containing 3�-overhangs from the single-strand RNA terminus (S)or the blunt terminus (B), resulting in cleavage within the adjacent 3� single-strand RNA region. The enzymedepicted is as described in the legend to Fig. 6. The pCp substitution interferes with the interaction of theenzyme at the terminus of the 3�-overhang, ablating cleavages generated from the 3� single-strand terminus(S). C, interaction of Dicer with substrates containing 5� single-strand RNA overhangs. Dicer cleaves the 23- and25-base pair substrates containing 5�-overhangs from the blunt terminus or overhang terminus. Dicer cleavesthe 17–21-base pair substrates containing 5�-overhangs exclusively from the blunt terminus, resulting in cleav-age within the adjacent 5� single-strand RNA region.




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appears to be due to both the preferential loading of the anti-sense strand into Ago2 and the enhanced binding affinity ofDicer for the siRNA, resulting in more siRNA loading intoRISC. Based on our model in which Dicer binding affinity forsiRNAs and ssRNAs is influenced predominantly by the PAZinteractionwith the 3� terminus of the strands of the duplex, thepredicted binding orientation for preferentially loading theantisense strand of the siRNA into RISC is with the PAZdomain of Dicer positioned at the 3� terminus of the antisensestrand (Fig. 7A). In the absence of 3�-overhang structures, thethermodynamic stability of the ends of the length-matchedsiRNA duplex would have a greater influence on this interac-tion, given that the PAZ interaction requires a free 3� terminus.Consequently, the less stable A:U base pairs would befavored over G:C base pairs. In addition, the binding affini-ties observed for ssRNA suggest that Dicer would preferen-tially bind the strand containing purine compared with py-rimidine residues at the 3� terminus. In fact, the HA-Ago2cleavage activity for the length-matched si92 duplex sug-gests that the sense strand containing adenine residues at the3� terminus was preferentially loaded into Ago2 over theantisense strand containing uridine residues at the 3� termi-nus (Fig. 6).The alternating 2�-fluoro/2�-methoxy-modified siRNAs

were previously shown to be more potent inhibitors of geneexpression comparedwith unmodified siRNAs (26). Consistentwith these observations, chemical modification of the siRNAswith alternating 2�-fluoro/2�-methoxy substitutions enhancedthe potency of the si92 and si97 siRNAs (Table 3). In addition,Dicer exhibited tighter binding affinities for the modifiedsiRNAs compared with the unmodified siRNAs (Table 3).Again, the enhancement in potency may be due to both tighterbinding affinity for Dicer and enhanced bias toward antisensestrand loading. Specifically, the rank order for binding to mod-ified ssRNAs was reversed, with tighter binding affinitiesobserved for the antisense strands of the si92 and si97 duplexescompared with the sense strands, which would bias the bindingdirectionality of Dicer to preferentially load the antisensestrands of themodified siRNAs into RISC (Table 3 and Fig. 7A).In the case of the si92 duplexes, the enhancement in bindingaffinity appeared to correlate with the enhancement in thepotency of themodified siRNAs comparedwith the unmodifiedduplexes (Table 3 (top)). The modified si97 duplexes, on theother hand, showed a significantly greater (20–30-fold)enhancement in potency comparedwith the 5–7-fold enhance-ment in binding affinity over the unmodified siRNAs (Table 3(bottom)). Given that the maximal enhancement in siRNAactivity due to preferential strand loadingwould be 2-fold, thesetwo factors alone cannot account for the significantly greaterimprovement in potency observed for the modified si97duplexes. Clearly, other factors are contributing to the activitiesof the siRNAs.In conclusion, our results demonstrate that Dicer can bind to

a wide range of oligoribonucleotides and that the affinitiesobserved are consistent with Dicer participating in both thecleavage of the siRNA precursors and the translocation of thesiRNA products to RISC.We demonstrate that the 3� terminusis crucial in binding interaction of the enzyme with the sub-

strate and that this process is sensitive to base composition.This may provide an explanation as to why in only some casesDicer substrates have been shown to be more potent than thecomparable siRNAs. Moreover, Dicer appears to be promiscu-ous in both binding and cleavage, since the enzyme is capable ofcleaving in double-strand and single-strand regions and canalso bind to single-strand regions and cleave in a duplex.Clearly, Dicer interacts with other proteins, such as TRBP andAgo2 (17). Future studies should address how these protein-protein interactions affect the binding and cleavage propertiesof Dicer.

REFERENCES1. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., andMello,

C. C. (1998) Nature 391, 806–8112. Bernstein, E., Caudy, A. A., Hammond, S. M., and Hannon, G. J. (2001)

Nature 409, 363–3663. Grishok, A., Pasquinelli, A. E., Conte, D., Li N., Parrish, S., Ha I., Baillie,

D. L., Fire, A., Ruvkun, G., and Mello, C. C. (2001) Cell 106, 23–244. Hutvagner, G., McLachlan, J., Pasquinelli, A. E., Balint, E., Tuschl, T., and

Zamore, P. D. (2001) Science 293, 834–8385. Ketting, R. F., Fischer, S. E., Bernstein, E., Sijen, T., Hannon, G. J., and

Plasterk, R. H. (2001) Genes Dev. 15, 2654–26596. Knight, S. W., and Bass, B. L. (2001) Science 293, 2269–22717. Zamore, P.D.,Tuschl,T., Sharp, P.A., andBartel,D. P. (2000)Cell101,25–338. Elbashir, S. M., Lendeckel, W., and Tuschl, T. (2001) Genes Dev. 15,

188–2009. Hammond, S. M., Bernstein, E., Beach, D., and Hannon, G. J. (2000) Na-

ture 404, 293–29610. Zhang, H., Kolb, F. A., Brondani, V., Billy, E., and Filipowicz, W. (2002)

EMBO J. 21, 5875–588511. Billy, E., Brondani, V., Zhang, H., Muller, U., and Filipowicz, W. (2001)

Proc. Natl. Acad. Sci. U. S. A., 98, 14428–1443312. Cerutti, L., Mian, N., and Bateman, A. (2000) Trends Biochem. Sci. 25,

481–48213. Zhang, H., Kolb, F. A., Jaskiewicz, L., Westhof, E., and Filipowicz, W.

(2004) Cell 118, 57–6814. MacRae, I. J., Zhou, K., Fei, L., Repic, A., Brooks, A. N., Cande, W. Z.,

Adams, P. D., and Doudna, J. A. (2006) Science 311, 195–19815. Sontheimer, E. J. (2005) Nat Rev. Mol. Cell. Biol. 6, 127–13816. Gatignol, A., Buckler-White, A., Berkhout, B., and Jeang, K. T. (1991)

Science 251, 1597–160017. Chendrimada, T. P., Gregory, R. I., Kumaraswamy, E., Norman, J., Cooch,

N., Nishikura, K., and Shiekhattar R. (2005) Nature 436, 740–74418. Doi, N., Zenno, S., Ueda, R., Ohki-Hamazaki, H., Ui-Tei, K., and Saigo, K.

(2003) Curr. Biol. 13, 41–4619. Vickers, T. A., Lima,W. F., Nichols, J. G., and Crooke, S. T. (2007)Nucleic

Acids Res. 35, 6598–661020. Kanellopoulou, C., Muljo, S. A., Kung, A. L., Ganesan, S., Drapkin, R.,

Jenuwein, T., Livingston, D.M., and Rajewsky, A. K. (2005)Genes Dev. 19,489–501

21. Kim, D. H., Behlke,M. A., Rose, S. D., Chang,M. S., Choi, S., and Rossi, J. J.(2005) Nat. Biotechnol. 23, 222–226

22. Tomari, Y., Matranga, C., Haley, B., Martinez, N., and Zamore, P. D.(2004) Science 306, 1377–1380

23. Rose, S. D., Kim,D.H., Amarzguioui,M., Heidel, J. D., Collingwood,M.A.,Davis, M. E., Rossi, J. J., and Behlke, M. A. (2005) Nucleic Acids Res. 33,4140–4156

24. Vermeulen, A., Behlen, L., Reynold,s A., Wolfson, A., Marshall, W. S.,Karpilow, J., and Khvorova, A. (2005) RNA 11, 674–682

25. Provost, P., Dishart, D., Doucet, J., Friendewey, D., Samuelsson, B., andRadmark, O. (2002) EMBO J. 21, 5864–5874

26. Allerson, C. R., Sioufi, N., Jarres, R., Prakash, T. P., Naik, N., Berdeja, A.,Wanders, L., Griffey, R. H., Swayze, E. E., and Bhat, B. (2005) J.Med. Chem.48, 901–904

27. Ausubel, F. M. (1988) in Current Protocols in Molecular Biology (Ausubel,




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A.,and Struhl, K., eds) p. 3.10.3, John Wiley & Sons, Inc., New York.

28. Lima, W. F., and Crooke, S. T. (1997) Biochemistry 36, 390–39829. Boulta, A., Delidakis, C., LivaDaras, I., and Tabler, M. (2003)Oligonucleo-

tides 13, 295–30130. Hohjoh, H. (2002) FEBS Lett. 521, 195–19931. Lingel, A., Simon, B., Izaurralde, E., and Sattler,M. (2004)Nat. Struct.Mol.

Biol. 11, 576–57732. Lee, H. S., Lee, S. N., Joo, C. H., Lee, H., Lee, H. S., Yoon, S. Y., Kim, Y. K.,

and Choe, H. (2007) J. Mol. Graph. Model. 25, 784–793.33. Song, J.-J., Liu, J., Tolia, N. H., Schneiderman, J., Smith, S. K., Martienssen,

R. A., Hannon, G. J., and Joshua-Tor, L. (2003) Nat. Struct. Biol. 10,1026–1032

34. Lima, W. F., and Crooke, S. T. (1997) J. Biol. Chem. 272, 27513–27516




http://ww

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Prakash, Andres R. Berdeja, Hans J. Gaus and Stanley T. CrookeWalt F. Lima, Heather Murray, Josh G. Nichols, Hongjiang Wu, Hong Sun, Thazha P.

Affinity and Interacts with Different Regions of the Nucleic AcidsHuman Dicer Binds Short Single-strand and Double-strand RNA with High

doi: 10.1074/jbc.M803748200 originally published online November 18, 20082009, 284:2535-2548.J. Biol. Chem.

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