Nature Protocols: doi: /nprot Supplementary Figure 1

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1 Supplementary Figure 1 Predicted secondary structures of the linear RCR template and elongated concatamer RCR product. (a) The linear RCR template is predicted to have a 3-way junction secondary structure. Sequence of template is shown in Table 1. (b) The elongated concatamer RCR product is predicted to have branched aptamer structures protruding and aligning on alternative sides and many dsdna (for drug loading) on the backbone and stem. Structures were predicted using the Nupack software.

2 Supplementary Figure 2 Electrophoresis and confocal fluorescence microscopy characterization of NFs. a) Agarose gel (2%) electrophoresis image indicating the elongation of DNA through RCR. Lane M: 20 bp DNA marker, lane 1: template, lane 2: primer, lane 3: template and primer after T4 DNA ligase treatment. The tailing results from the complicated structures formed through intra- and intermolecular base pairs. Lane 4: RCR product after phi29 DNA polymerase treatment. Polymerization of dntp monomers and dense packaging-driven assembly results in huge molecular weight of RCR products; therefore, the corresponding band shows no migration. Hu, R. et al. DNA nanoflowers for multiplexed cellular imaging and traceable targeted drug delivery. Angew. Chem. Int. Ed Volume 53. Pages Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Adapted with permission. b) Confocal fluorescence microscopy imaging of Cy5-dUTP-integrated NFs. The fluorescence signal (Red) of Cy5 (maximum excitation wavelength is 650 nm and maximum emission wavelength is 662 nm) is obvious and indicates the successful polymerization of Cy5-dUTP and the normal function of Cy5. NFs with a size of 2 μm (RCR for h) are used for this imaging. Scale bar, 5 μm. Adapted with permission from J. Am. Chem. Soc. 2013, 135(44), Copyright 2013 American Chemical Society.

3 Supplementary Figure 3 Characterizations of NFs. a) Dynamic light scattering measurement of NFs. DLS data reveals the size distribution of NFs and the average radius is calculated to be about 150 nm. NFs with 15 h of RCR reaction time are used in this experiment. Hu, R. et al. DNA nanoflowers for multiplexed cellular imaging and traceable targeted drug delivery. Angew. Chem. Int. Ed Volume 53. Pages Copyright Wiley- VCH Verlag GmbH & Co. KGaA. Adapted with permission. b) TEM images of NFs displays ultrathin sheet sections (indicated by arrows). c) Enlarged partial view of the dashed box. These results provide clear evidence of the internal hierarchical structures and the dense DNA packaging in NFs. NFs with 10 h of RCR reaction time are used for this experiment. Adapted from ref. 31. d) AFM imaging of NFs. e) Cross-sectional plot indicated with the white line. AFM imaging displays that the NFs are monodisperse with a size of nm. NFs with 10 h of RCR reaction time are used for this experiment. Adapted from ref. 31. f) Bright field. g) Polarized light imaging of (f). When exposed between crossed polarizers, NFs displayed disc-shaped optical textures as a result of the birefringence of liquid crystalline structure, which is an optical property of anisotropic materials. This is a direct demonstration of liquid crystalline structures of NFs. NFs with h of RCR reaction time are used for this experiment. Panels b g adapted with permission from J. Am. Chem. Soc. 2013, 135(44), Copyright 2013 American Chemical Society.

4 Supplementary Figure 4 Exceptional stability of NFs. (a c) SEM images of NFs treated with DNase I (a, b, 5 U/mL) and human serum (c, 10% diluted) for 24 h at 37 C. (d f) SEM images displaying NFs heated at 170 C for 0.5 h (d), treated with urea (5 M) for 0.5 h (e), and diluted 100 times (f). The maintenance of NF structural integrity indicates the high stability of NFs. Adapted with permission from J. Am. Chem. Soc. 2013, 135(44), Copyright 2013 American Chemical Society.

5 Supplementary Figure 5 Flow cytometry results demonstrating the selective recognition of sgc8c-incorporated NFs. (a) CEM cells (target cell line, suspension); dark green=cell only, light green=cells incubated with NFs. (b) Ramos cells (nontarget cell line, suspension); dark green=cell only, light green=cells incubated with NFs. (c) HeLa cells (target cell line, adherent); gray=cell only, red=cells incubated with NFs. Target cell lines show an obvious fluorescence signal shift compared with nontarget cell line, which demonstrates a high selectivity of NFs toward target cells. These results show that the conjugated aptamer preserves its binding affinity and specificity. NFs with a size of 200~300 nm (RCR for h) are used for this experiment. Angew. Chem. Int. Ed Volume 53. Pages Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Adapted with permission.

6 Supplementary Figure 6 Drug-loading into the preferable drug-associated sequences of NFs and the cytotoxicity of NFs toward target/nontarget cells. (a) Drug-loading into the preferable drug-associated sequences of NFs. Left: NFs are incubated with Dox in Dulbecco s PBS at room temperature. The whole mixture shows uniform red, which is the color of Dox solution. Right: After incubation for 24 hours, the mixture is centrifuged at 14,000 g for 5 min. The supernatant turns light upon losing part of the Dox, while the NF concentrate turns dark red upon Dox loading. Both observation with the naked eye and the following ultraviolet absorption measurement demonstrate the drugloading ability of DNA NFs. NFs with a size of 200~300 nm (RCR for h) are used for this experiment. (b) The correspondence between NF concentration and cell viability. Red line, cell viability of target CEM cells with increasing NF concentration; black line, cell viability of nonarget Ramos cells with increasing NF concentration. No significant cytotoxicity of NFs toward either target or nontarget cells is observed in panel a, which indicates the satisfactory biocompatibility of NFs. (c) SEM images of NFs used in this cytotoxicity experiment. Scale bar: 50 μm. Inset panel, high-resolution imaging of a single NF. Scale bar: 300 nm. NFs with a size of 200~300 nm (RCR for h) are used for this experiment. Panels b and c adapted with permission from J. Am. Chem. Soc. 2013, 135(44), Copyright 2013 American Chemical Society.

7 Supplementary Figure 7 MTS assay results show selective cytotoxicity of Dox delivered by NFs. (a) Cell viability of target HeLa cells with NF-Dox complex or free Dox treatment. (b) Cell viability of target CEM cells with NF-Dox complex or free Dox treatment. (c) Cell viability of nontarget Ramos cells with NF-Dox complex or free Dox treatment. Red triangle, cytotoxicity of free Dox; black dots, cytotoxicity of NF-Dox complex. Free Dox shows nonselectivity toward both target (HeLa and CEM cells) and nontarget (Ramos cells) cell lines, while NF-Dox complex only shows cytotoxicity toward target cell lines (HeLa and CEM cells). In contrast to nonselective cytotoxicity of free Dox in both target cells and nontarget cells, the selective cytotoxicity of Dox delivered by NFs indicates their ability to perform targeted drug delivery. NFs with a size of 200~300 nm (RCR for h) were used for this experiment. Adapted with permission from J. Am. Chem. Soc. 2013, 135(44), Copyright 2013 American Chemical Society.

8 Supplementary Method For those who will make their own DNAs with a synthesizer, we provide suggested steps consisting of DNA synthesis, DNA deprotection and purification, DNA desalting, and determination of DNA concentration. DNA synthesis TIMING ~1 d S1 Equip the DNA synthesizer with vials of solutions of conventional da, dg, dc, and dt, as well as a vial containing the solution of CPR II. S2 Set the da-cpg support (for primer) and dt-cpg support (for template) on the DNA synthesizer and start the synthesis at a scale of 1.0 μmol with the DMT-on program. See Table 1 for sequences mentioned above. CRITICAL STEP During synthesis, the ambient temperature should be kept between 18 to 23 C, and humidity should be kept as low as possible. CPR II is stable in solution, but only for 2-3 days, while conventional da, dg, dc, and dt phosphoramidites are stable in solution for ~1 week.! CAUTION All reagents used in DNA synthesis are potentially harmful, and some of them are corrosive and volatile (see REAGENTS for details). DNA deprotection and purification TIMING 1-2 d S3 After synthesis, dry the column with nitrogen, and transfer the CPG supports to a 15 ml centrifuge tube with screw cap. CRITICAL STEP Centrifuge tube with screw cap could effectively prevent steam burst caused by heated volatilization in the next step. S4 Use AMA for deprotection following instructions from the manufacturer.! CAUTION AMA is volatile. S5 Wait until the deprotection solution cools down. Use a pipette to transfer the supernatant into a plastic centrifuge tube. CRITICAL STEP In this step, try to leave as many CPG beads behind as possible. The remaining few beads will be filtered out before HPLC purification. S6 To each tube, add 200 μl of 3 M NaCl and 5 ml of cold ethanol. Mix by vortexing. Salts and DNAs will precipitate as floccule. S7 Freeze for at least 30 minutes.

9 PAUSE POINT For complete precipitation, an entire day can be taken for the freezing step. S8 Centrifuge for minutes at 14,000g and 4 C. S9 Gently discard supernatant and keep the deposition in the bottom and inside wall of the tube. S10 Add 400 μl of 0.1 M TEAA to each tube and dissolve the deposition with a pipette tip. S11 Transfer the solution to 2 ml centrifuge tubes individually and centrifuge for 1 minute at 14,000g. S12 Filter the supernatant with micro-pore membrane to remove residual beads and salt deposition. PAUSE POINT If necessary, samples in this step can be frozen at -20 C and kept for a few months. S13 Connect the C-18 reversed-phase column to HPLC. S14 Purify DNA sample with HPLC (See EQUIPMENT SETUP). S15 Collect the DNA fraction using 2 ml centrifuge tubes according to product peak. CRITICAL STEP In an efficient synthesis, the main peak usually belongs to the products, while the retention effect of DMT groups has the longest retention time. S16 Use a centrifugal vacuum evaporator to remove solvent. PAUSE POINT Overnight evaporation is typically needed, and dark treatment is strongly suggested during evaporation in case of potential photodamage. After drying the solutions, the dried DNA samples can be resolved with 500 μl of TEAA and steps 13 to 16 repeated, if highly purified DNA products are desired. If the samples are pure enough, keep them dry for further steps. S17 For primer, add 200 μl of 80% acetic acid in total to dissolve each dried sample and incubate for 20 min at room temperature. Then add 500 μl of cold ethanol and 20 μl of 3 M NaCl. Vortex and freeze at -20 C. Primers will participate as floccule. Centrifuge for 5 min at 14,000g. Gently discard supernatant and keep the deposition at the bottom and inside wall of the tube. Use a centrifugal vacuum evaporator to remove

10 solvent. Evaporation for 30 min is usually sufficient. S18 For template samples, add 200 μl of 20% acetic acid in total to dissolve each dried sample and incubate for 1 h at room temperature. Dry the oligonucleotide down and add ammonium hydroxide. Leave at room temperature for 15 minutes to achieve complete elimination of the side chain to the 5 -phosphate. Use a centrifugal vacuum evaporator to remove solvent. DNA desalting TIMING ~2 h S19 Dissolve the dried samples with 500 μl of ultrapure water and vortex for thorough mixing. S20 Set up a Nap-5 desalting column and remove the top and bottom caps from the column. S21 Wash the column with more than 15 ml of ultrapure water. Allow the water to completely enter the gel bed by gravity flow. No collection is needed in this step. CRITICAL STEP Do not apply positive pressure. S22 Add DNA solution from step S19 (500 μl in total) to the column. Allow the sample to completely enter the gel bed. No collection is needed in this step. S23 Place a 2 ml centrifuge tube under the column. S24 Add 1 ml of ultrapure water to the column and elute the purified sample. S25 Repeat steps S21 to S24 for desalting of other DNA samples. CRITICAL STEP Two desalting columns can be used at the same time to save time. After use, column(s) should be washed with more than 10 ml of ultrapure water. Cap the column(s) on the top and bottom and store at room temperature. Please follow the instructions from manufacturer for more information about the Nap-5 desalting column. Determination of DNA concentration TIMING ~30min S26 Use a UV-visible spectrometer to measure the absorbance of the collected solution at 260 nm. When the absorbance exceeds maximum detectability, adequately dilute the solution with ultrapure water. S27 Calculate the concentration of DNA samples according to the following formula: c=a/(εb), where c is the concentration of DNA sample, A is the absorbance at 260 nm, d is the thickness of the cell (cm), and ε is the molar extinction coefficient of

11 the native DNA with the same sequence. Continue with the procedure from Step 2. CRITICAL STEP Molar extinction coefficient (ε) of the native DNA can be calculated using such open access software as Oligo Analyzer from IDT ( 3 or 5 modifications should be considered when performing calculations.