Supporting InformationVenkataraman et al. 10.1073/pnas.1006377107SI TextMethods. Marker selection and hairpin sequence design.Each cancermarker is an 18-nt mRNA segment spanning the splice point of amutant fusion transcript. Each HCR hairpin has a 4-nt toehold, a14-bp stem, and a 4-nt hairpin loop. With the exception of theloop of hairpin A and the complementary toehold of hairpin B,the hairpin sequences are fully determined by the sequence of themarker. For each mutant fusion, we considered different markerplacements such that the splice point was positioned within 3-ntof the center of the marker. Furthermore, for each marker place-ment, we considered all possible (44 ! 256) hairpin sequencedesigns. Candidate marker placements and hairpin designs wereevaluated based on thermodynamic criteria using NUPACK(www.nupack.org) (1). For a given target secondary structure,the ensemble defect represents the average number of incorrectlypaired nucleotides at equilibrium, calculated over the ensembleof unpseudoknotted secondary structures (2). We evaluated can-didate sequences by calculating the sum of the ensemble defectsover four target secondary structures (IA, A, IB, B). Here, IAdenotes the (unstructured) input to hairpin A (correspondingto the marker candidate M, as well as the output domain thatis exposed upon opening hairpin B) and IB denotes the (unstruc-tured) input to hairpin B (corresponding to the output domainthat is exposed upon opening hairpin A).

Selective polymerization assay. Oligonucleotides were synthesizedand HPLC-purified by Integrated DNA Technologies. Stock so-lutions of each oligonucleotide (1.25 !M markers and 2.5 !Mhairpins) were prepared in 1 ! PKR buffer (20 mM Hepes pH7.5, 4 mMMgCl2, 100 mM KCl). The solutions were snap-cooled(heat at 95 °C for 90 sec, cool rapidly on ice and then leave atroom temperature for 30 min) and then mixed to react at thefollowing concentrations in 1 ! PKR buffer: 0.4 !M (5.2 pmol)for each marker and 0.8 !M (10.4 pmol) for each hairpin (totalvolume: 13 !L). Reactions were allowed to proceed at 37 °C for30 min, then supplemented with 3.25 !L of 5 ! native loadingbuffer (50% glycerol (v!v), bromophenol blue and xylene cyanol)and run on 10% TBE (89 mM Tris/89 mM boric acid/2 mMEDTA, pH 8.3) native polyacrylamide gels for 60 min at100 V. Gels were stained using SYBR Gold (Invitrogen;S11494) and imaged using an FLA-5100 fluorescent scanner(Fujifilm Life Science).

Selective PKR activation assay. PKR was expressed and purified asdescribed by Zheng and Bevilacqua (3). 50 pmol of PKR werefirst dephosphorylated using " protein phosphatase (New Eng-land Biolabs; P0753L). The " phosphatase was inactivated bytreatment with freshly prepared sodium orthovanadate as de-scribed by Matsui et al. (4). Stock solutions of each oligonucleo-tide (0.4 !M markers and 0.8 !M for each hairpin) were snap-cooled as described earlier and then reactants were mixed in 1 !PKR buffer: 4 pmol of PKR, 1 nmol of ATP, 0.8 pmol of marker,1.6 pmol of each hairpin (total reaction volume: 20 !L). Reac-tions were allowed to proceed for 30 min at 30 °C and thenstopped by adding 20 !L of 2 ! reducing SDS loading buffer(25% glycerol, 7.5% SDS (w!v), 150 mM Tris-HCl (pH 6.8 at25 °C), 10 mg bromophenol blue and 50 mM DTTadded fresh),denatured by heating at 90 °C for 5 min, and allowed to cool atroom temperature for 5 min. 25 !L of each reaction were run on10% SDS polyacrylamide gels for 40 min at 150 V. PKRwas trans-

ferred to nitrocellulose membranes (Invitrogen; IB301001)using an iBlot device (Invitrogen). The membranes were probedfor PKR phosphorylated at Thr-451 as described below (Wes-tern blots).

Cell culture. The glioblastoma cell lines U87MG-#EGFR (5) andU87MG-wt EGFR (6) were received as a gift from F. Furnari(Ludwig Institute for Cancer Research, San Diego). Cells werepropagated in DMEM (Invitrogen; 11885-092) supplementedwith 10% FBS and 400 !g!mL (50% activity) G418 selectionantibiotic. The prostate carcinoma cell line, LNCaP (7), was pur-chased from the American Type Culture Collection (ATCC)(ATCC number: CRL-1740). Cells were propagated in RPMImedium 1640 (Invitrogen; A10491-01) supplemented with 10%FBS. The Ewing’s sarcoma cell line, TC71(8, 9), was receivedas a gift from M.E. Davis (California Institute of Technology,Pasadena, USA). Cells were propagated in RPMI medium1640 supplemented with 10% FBS. All cells were propagatedin the presence of 100 U!mL of penicillin and 100 !g!mL ofstreptomycin.

Transfection. 50 !M stock solutions of each oligonucleotide wereprepared in 1 ! PKR buffer and snap-cooled. Hairpin solutionswere mixed prior to transfection so that the total concentration ofRNA in solution was 100 nM (using equimolar amounts of Aand B hairpin for solutions containing both). U87MG-#EGFRand U87MG-wtEGFR cells were seeded into 12-well plates ata density of 20,000 cells in 1 mL of medium per well and grownovernight. Prior to transfection, cells were washed with warmPBS, and fresh medium containing no antibiotics or serum wasadded. U87MG-#EGFR and U87MG-wtEGFR cells were trans-fected using Oligofectamine (Invitrogen; 12252-011) according tothe manufacturer’s instructions, using 1.5 !L of Oligofectamineper well. LNCaP and TC71 cells were seeded into 12-well platesat a density of 200,000 cells in 1 mL of medium per well. Trans-fection was performed using HiPerFect (Qiagen; 301705) accord-ing to the manufacturer’s Fast-Forward transfection protocol,using 12 !L of HiPerFect per well.

Cell survival assays. Twenty hours posttransfection, cells wereimaged using an inverted microscope in phase contrast mode(Zeiss Axio Observer.Z1) with an Enhanced Contrast (EC)Plan-Neofluar 10 ! !0.30 objective (Zeiss; 440330-9902-000).Cells were then dislodged from the surface using trypsin, pelleted,and each sample was resuspended in 200 !L of fresh media sup-plemented with 0.1% sodium azide (w!v) (a preservative) and2 !g!mL 7-aminoactinomycin D (7-AAD) (an exclusion dye usedto test for plasma membrane integrity) (Invitrogen; A1310). Thesurviving cells were counted by flow cytometery (Beckman Coul-ter Quanta SC MPL or Accuri C6). Viable cells were identifiedusing three gates: forward scatter, side scatter, and low 7-AADfluorescence. Appropriate gates were determined using an un-treated sample of cells. For each type of treated or mock experi-ment, we calculate the mean, !, and sample standard deviation, $,over six independent cell samples. The fraction of survivingcells is reported as a fractional mean, !fractional ! !treated!!mock,with error bars representing fractional standard deviation (10),"fractional ! !fractional"#"mock!!mock$2 % #"treated!!treated$2&1!2.

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Regrowth experiments. Cells were plated and transfected as de-scribed above. For each cell line, six wells were mock transfectedand twelve wells were transfected with the appropriate HCRtransducer. 20 hr posttreatment, the mock transfections and sixof the HCR transfections were imaged and analyzed by flow cyto-metry as described above. The remaining six HCR transfectionswereallowedtogrowuntil theyreachedconfluency.Theywerethendislodged from the surface using trypsin, combined and reseededinto twelve wells at the original densities (20,000 cells per mL forU87MG-#EGFRandU87MG-wtEGFR; 200,000 cells permL forLNCaPandTC71). The cells were then transfected again: sixmocktransfections and six HCR transfections. Again, 20 hr posttreat-ment, the cells were imaged and analyzed by flow cytometry.

PKR inhibition. PKR was inhibited in cell culture by supplementingthe media with 5 mM 2-aminopurine (2-AP) (MP Biomedicals;159862). For U87MG-#EGFR and U87MG-wtEGFR cells,2-AP was added to the media two hours prior to transfection.For LNCaP and TC71 cells, 2-AP was added to the media at thetime of seeding the cells into 12-well plates.

Western blots.Approximately 1 ! 106 cells were pelleted and lysedwith 150 !L hypotonic lysis buffer (20 mM Hepes, 20 mM NaCl,5 mM EDTA, 0.1% Tween 20, protease inhibitor cocktail [RocheApplied Science; 04693116001], phosphatase inhibitor cocktails[Sigma-Aldrich; P2850 and P5726]) for 10 min at room tempera-ture. The lysates were centrifuged at 16;000 ! g for 10 min at 4 °C,afterwhich the supernatantswere collected. Protein concentrationwas determined using a Bradford assay (Sigma-Aldrich; B6916).For each sample, 25 !g of lysate were denatured and run on10% Tris -HCl polyacry lamide gels in 1 ! Tris!Glycine!SDS running buffer (Biorad; 161-0732). SeeBlue Plus2 andMagicMark XP (Invitrogen; LC5925 and LC5602) size standardswere also run on the gels. The proteins were transferred to nitro-cellulose membranes (Invitrogen; IB301001) using an iBlotdevice (Invitrogen). The membranes were blocked overnight at4 °C using 5% BSA (w!v) (Sigma-Aldrich; A4503) in 1 ! TBST(20 mMTris-HCl pH 7.5, 0.9%NaCl, 0.1% Tween 20). The block-ing solution was decanted and the membranes were then probedwith one of the following primary antibodies for 3 h: rabbit poly-clonal to phosphorylated PKR (phospho T-451; Abcam; ab4818)or rabbitmonoclonal to phosphorylated eIF2% (phospho S-51;Ab-cam; ab32157), diluted 1,000-fold or 500-fold in 1 ! TBST, respec-tively. The membranes were washed four times with 1 ! TBST for5 min per wash. An enhanced chemiluminescence (ECL) anti-rabbit horseradish perioxidase (HRP)-linked secondary antibody(GE Healthcare Life Sciences; NA934) was used for detection(diluted 2,000-fold in 1 ! TBST and incubated for 1 hr). Themembranes were then washed for 1 h with 1 ! TBST, followedby three washes with 1 ! TBST for 5 min each. SuperSignal WestPico chemiluminescent substrate (Pierce Protein Research;PI34077) was then added to eachmembrane and allowed to devel-op for 3 min. Excess liquid was dripped off and the blots were im-aged using KodakX-OMATXAR-5 film (VWR; IB1651496). Theblots were then stripped by incubating with mild stripping buffer(to make 1 L: 15 g glycine, 1 g SDS, 10 mLTween 20, adjust pH to2.2, add ultrapure water to 1 L) for 5–10 min. The buffer was dis-carded and fresh stripping buffer was added for another 5–10min.The following washes were then performed: two times 10 min in1 ! PBS (phosphate buffered saline) and two times 5 min in1 ! TBST (using enough to cover themembrane). Themembraneswere reprobed for &-actin as a loading control, using a rabbitpolyclonal antibody (Abcam; ab8227). The blots were performedusing a SNAP i.d. protein detection system (Millipore) accordingto the manufacturer’s protocol. The blots were blocked using 3%BSA in 1 ! TBST.

DNA fragmentation assays. Approximately 2 ! 106 cells werepelleted 4 h after treatment. Chromosomal DNA was extractedusing the Apoptotic DNA Ladder Kit (Roche; 11835246001)according to the manufacturer’s instructions. 2 !g of each samplewere run (45 min at 150 V) on 1% agarose gels made with lithiumboric acid (LB) buffer (Faster Better Media LLC; LB20-10) andcontaining ethidium bromide (200 ng!mL). The gels were visua-lized using an UV gel imager (Alpha Innotech AlphaImager).Lane 1 contains a DNA ladder (New England BioLabs; N3231).

Marker sequences. The cancer markers are 18-nt subsequences offusion transcripts that appear in human cancer cell lines (splicepoint underscored):

M1: 5!-AAA AGA AAG GUA AUU AUG-3!M2: 5!-AAG AAG AAA UAU GUG GGA-3!M3: 5!-AGC AGA ACC CUU CUU AUG-3!

M1 is the #egfr fusion resulting from deletion of exons 2–7 in theepidermal growth factor receptor gene, commonly found in glio-blastomas, including the glioblastoma cell line U87MG-#EGFR(5), and also reported in breast, ovarian, prostate, and lung car-cinomas (11). M2 is the tpc/hpr fusion resulting from transloca-tion t(6;16)(p21;q22) found in the prostate cancer cell lineLNCaP (7). M3 is the ews/fli1 fusion resulting from translocationt(11;22)(q24;q12) found in the Ewing’s sarcoma cell line TC71 (8,9) and present in 85% of Ewing’s family tumors (12).

The 5! and 3! wild-type subsequences that are disrupted by thefusion are as follows (underscores denote subsequences thatappear in the fusion):

M1-wt5!: 5!-AAA AGA AAG UUU GCC AAG-3!M1-wt3!: 5!-AGU GUC CCC GUA AUU AUG-3!M2-wt5!: 5!-AAG AAG AAA GCA AGU CAU-3!M2-wt3!: 5!-GUG AAG CAG UAU GUG GGA-3!M3-wt5!: 5!-AGC AGA GUU CAU UCC UUC-3!M3-wt3!: 5!-AAG AAG ACC CUU CUU AUG-3!.

The marker (toehold underscored):M4: 5!-ACA AAA AUU UGU GCU AUG-3!

is a subsequence of the egfr and #egfr transcripts with the sametoehold sequence as marker M1. M4 is used both as a positivecontrol for HCR-mediated cell death in selectivity studies usingU87MG-wtEGFR cells and for HCR mechanism studies withU87MG-#EGFRcells.

HCR hairpin sequences. Hairpins have 4-nt toeholds/loops and14-bp duplex stems. The following HCR transducers acceptmRNA cancer markers as inputs (toehold underscored):

Transducer HCR1.A1: 5!-CAU AAU UAC CUU UCU UUU GGG CAA AAG

AAA GGU AAU-3!B1: 5!-AAA AGA AAG GUA AUU AUG AUU ACC UUU

CUU UUG CCC-3!Transducer HCR2.A2: 5!-UCC CAC AUA UUU CUU CUU GAG UAA GAA

GAA AUA UGU-3!B2: 5!-AAG AAG AAA UAU GUG GGA ACA UAU UUC

UUC UUA CUC-3!Transducer HCR3.A3: 5!-CAG ACA UAA GAA GGG UUC AGC AGA ACC

CUU CUU AUG-3!B3: 5!-GAA CCC UUC UUA UGU CUG CAU AAG AAG

GGU UCU GCU-3!The following hairpins are used in selectivity and mechanism

studies (underscores denote domain modifications with respectto HCR1):

Transducer HCR4.A4: 5!-CAU AGC ACA AAU UUU UGU GGG CAC AAA

AAU UUG UGC-3! = A1(#stem)

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B4: 5!-ACA AAA AUU UGU GCU AUG GCA CAA AUUUUU GUG CCC-3! = B1(#stem)

Transducer HCR5.A5: 5!-CAU AAU UAC CUU UCU UUU GCC CAA AAG

AAA GGU AAU-3! = A1(#loop)

B5: 5!-AAA AGA AAG GUA AUU AUG AUU ACC UUUCUU UUG GGC-3! = B1(#toehold)

B6: 5!-AAA AGA AAG GUA AUA AGU AUU ACC UUUCUU UUG CCC-3! = B1(#loop)

1. Zadeh JN, et al. (in press) NUPACK: analysis and design of nucleic acid systems. J Com-put Chem.

2. Dirks RM, Lin M, Winfree E, Pierce NA (2004) Paradigms for computational nucleic aciddesign. Nucleic Acids Res 32:1392–1403.

3. Zheng X, Bevilacqua PC (2004) Activation of the protein kinase PKR by short double-stranded RNAs with single-stranded tails. RNA 10:1934–1945.

4. Matsui T, Tanihara K, Date T (2001) Expression of unphosphorylated form of humandouble-stranded RNA-activated protein kinase in Escherichia coli. Biochem BiophysRes Commun 284:798–807.

5. Nishikawa R, et al. (1994) A mutant epidermal growth-factor receptor common in hu-man glioma confers enhanced tumorigenicity. Proc Natl Acad Sci USA 91:7727–7731.

6. Nagane M, et al. (1996) A common mutant epidermal growth factor receptor confersenhanced tumorigenicity on human glioblastoma cells by increasing proliferation andreducing apoptosis. Cancer Res 56:5079–5086.

7. Veronese ML, Bullrich F, Negrini M, Croce CM (1996) The t(6;16)(p21;q22) chromosometranslocation in the LNCaP prostate carcinoma cell line results in a tpc/hpr fusion gene.Cancer Res 56:728–732.

8. Whang-Peng J, et al. (1984) Chromosome translocation in peripheral neuroepithelio-ma. New Engl J Med 311:584–585.

9. Hu-Lieskovan S, Heidel JD, Bartlett DW, Davis ME, Triche TJ (2005) Sequence-specificknockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNAinhibits tumor growth in a murine model of metastatic Ewing’s sarcoma. CancerRes 65:8984–8992.

10. Taylor JR (1997) An introduction to error analysis: the study of uncertainties in physicalmeasurements (University Science Books, Sausalito, CA).

11. Kuan CT, Wikstrand CJ, Bigner DD (2001) EGF mutant receptor vIII as a moleculartarget in cancer therapy. Endocr-Relat Cancer 82:83–96.

12. Arvand A, Denny CT (2001) Biology of EWS/ETS fusions in Ewing’s family tumors.Oncogene 20:5747–5754.

Fig. S1. Additional control lanes for the conditional polymerization studies of Figs 2A and 5A reveal the mobility of each of the hairpins and markers incomparison to a dsRNA ladder. Native polyacrylamide gel electrophoresis.

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Fig. S2. Additional data for cell survival studies of Fig. 3. Absence of either hairpin species prevents polymerization, allowing cells to survive. Cell survival wasassayed 20 hr posttransfection. (A) Micrographs of surviving cell populations. (Scale bar: 100 !m). (B) Cell counts by flow cytometry. Normalized means over sixsets of experiments; error bars depict sample standard deviations.

Fig. S3. Transfection concentration study for the HCR hairpins and cell lines of Fig. 3. The efficacy of HCR decreases for RNA transfection concentrations below100 nM (50 nM for each hairpin species). Cell survival was assayed 20 hr posttransfection by flow cytometry. Normalized means over six sets of experiments;error bars depict sample standard deviations. As a point of reference, therapeutic RNAi studies that employed 100 nM siRNA transfections for cultured cellexperiments demonstrated that these siRNAs effectively inhibit tumor growth in mouse models for cancer (9, 1).

1 Takei Y, Kadomatsu K, Yuzawa Y, Matsuo S, & Muramatsu T (2004) A small interfering RNA targeting vascular endothelial growth factor as cancer therapeutics. Cancer Res 64(10):3365–3370.

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Fig. S4. Additional data for Fig. 4 efficacy study on cells that survive initial treatment. Micrographs of surviving cell populations. (Scale bar: 100 !m.) Cells weretransfected with cognate HCR transducers and survival was assayed 20 h posttransfection. After growing up surviving cells, cells were transfected a second timewith cognate HCR transducers and assayed for survival 20 h posttransfection.

BA

Fig. S5. Additional data for the mechanism studies of Fig. 5. Treatment with 2-AP, a PKR inhibitor, blocks cell death. Cell survival was assayed 20 hr post-transfection. (A) Micrographs of surviving cell populations. (Scale bar: 100 !m). (B) Cell counts by flow cytometry. Normalized means over six sets of experi-ments; error bars depict sample standard deviations.

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Fig. S6. Additional data for PKR inhibition controls of Figs 6A and B. Treatment with 2-AP, a PKR inhibitor, blocks cell death. Cell survival was assayed 20 hposttransfection. Micrographs of surviving cell populations. (Scale bar: 100 !m.)

Fig. S7. Caspase activation study. Apoptosis mediated by PKR is known to include activation of the caspase 8 and 9 pathways acting on caspase 3 (1). To assayfor selective caspase activation, we performed flow cytometry on cells treated with a fluorescently labeled broad-spectrum caspase inhibitor that selectivelybinds activated caspases. Elevated caspase activity is observed under the same conditions that yield DNA laddering in Fig. 6C. 4 h posttransfection, cells weredislodged from the surface using trypsin and pelleted. Cells were then stained with VAD-fmk conjugated to FITC (Biovision; K180) according to the manu-facturer’s instructions. Following staining, cells were analyzed by flow cytometery (Beckman Coulter Quanta SC MPL, BD FACSCalibur, or Accuri C6). Cells werepreserved by supplementing all staining and washing solutions with 0.1% sodium azide (w!v).

1 Garcia MA, Meurs EF, & Esteban M (2007) The dsRNA protein kinase PKR: virus and cell control. Biochimie 89:799–811.

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