Supporting Information

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1 Supporting Information Transfection of DNA Cages into Mammalian Cells Table of Contents Supporting Figure 1 DNA tetrahedra used in transfection experiments 2 Supporting Figure 2 Time courses of intracellular distributions of tetrahedra transfected with and without Lipofectin 3 Supporting Figure 3 Representative cell count histograms for data shown in Figure 1 4 Supporting Figure 4 Detection of transfection by biotin-labeled tetrahedra 5 Supporting Figure 5 DNA complexes used in control transfection experiments 7 Supporting Figure 6 Comparison between intracellular distributions of fluorescently labeled DNA complexes used for transfection 8 Supporting Figure 7 Confocal micrographs showing the photobleaching of an ROI 9 Supporting Figure 8 Supporting Figure 9 Supporting Figure 10 Intracellular localization of transfected Cy5 labeled DNA tetrahedra (additional time points for Figure 4) Intracellular localization of transfected Cy5 labeled DNA tetrahedra (additional time points for Figure 5) Representative confocal micrographs of fixed cells transfected with fluorescently labeled DNA tetrahedra

2 Modification Sequences 5 3 Structure i) Cy3 / Cy5 S1 Cy3 AGG CAG TTG AGA CGA ACA TTC CTA AGT CTG AAA TTT ATC ACC CGC CAT AGT AGA CGT ATC ACC S2 CTT GCT ACA CGA TTC AGA CTT AGG AAT GTT CGA CAT GCG AGG GTC CAA TAC CGA CGA TTA CAG S3 GGT GAT AAAACG TGT AGC AAG CTG TAA TCG ACG GGA AGA GCA TGC CCA TCC ACT ACT ATG GCG S4 Cy5 CCT CGC ATG ACT CAA CTG CCT GGT GAT ACG AGG ATG GGC ATG CTC TTC CCG ACG GTA TTG GAC ii) biotin S1Biotin AGG CAG TTG AGA CGA ACA TTC CTA AGT CTG AAA TTT ATC ACC CGC CAT AGT AGA CGT ATC ACC S2 CTT GCT ACA CGA TTC AGA CTT AGG AAT GTT CGA CAT GCG AGG GTC CAA TAC CGA CGA TTA CAG S3 GGT GAT AAAACG TGT AGC AAG CTG TAA TCG ACG GGA AGA GCA TGC CCA TCC ACT ACT ATG GCG S4 CCT CGC ATG ACT CAA CTG CCT GGT GAT ACG AGG ATG GGC ATG CTC TTC CCG ACG GTA TTG GAC Supporting Figure 1: DNA tetrahedra used in transfection experiments. Three types of fluorescent tetrahedra, labeled with Cy 3, Cy 5 or both Cy3 and Cy5 were prepared. Tetrahedra containing a single covalently attached biotin molecule were also prepared. 2

3 6hr With Lipofectin Without Lipofectin 24hr hr hr Neg. Control (24hr) 2 Supporting Figure 2: Time course of intracellular distributions of tetrahedra transfected with and without Lipofectin. Confocal fluorescence (red) and phase contrast images are superimposed. Mean cell fluorescence readings were determined by flow cytometry. Negative Control samples (Neg. Control) were transfected with unlabeled DNA tetrahedra. All other samples were transfected with DNA tetrahedra labeled with Cy5. Scale bars 100 μm. 3

4 6 hr with Lipofectin 72 hr with Lipofectin Supporting Figure 3: Representative cell count histograms for data shown in Figure 1. 4

5 Supporting Figure 4: Detection of transfection by biotin-labeled tetrahedra. Total DNA extracted from cell samples was bound to a positively charged nylon membrane. Biotin was detected using a kit incorporating streptavidin-linked alkaline phosphatase and a chemiluminescent substrate: chemiluminescence, induced by enzyme bound to the membrane though streptavidin-biotin interaction and recorded on photographic film, provides a measure of the amount of biotin in the sample. See below for a detailed description of methods. Row 1: (Control) Samples of biotin-labeled DNA tetrahedra which were applied directly onto the membrane immediately before detection. Dots a, b, c and d contained 42ng, 28ng, 14ng and 7ng of biotin-labeled tetrahedra respectively. Row 2: Samples of DNA extracted from cells which had been transfected with biotin labeled DNA tetrahedra without the aid of lipofectin. Dots a, b, c and d contained DNA extracted from cells 6hrs, 24hrs, 48hrs and 72hrs post transfection respectively. Each dot contained approximately 1/3 of the total DNA extracted from the sample. Row3: As row 2 except that cells were transfected with biotin labeled DNA tetrahedra with the aid of 5

6 lipofectin. Row4: (Control) As rows 2 except that cells were mock transfected without biotin labeled DNA cages. Each sample of transfected cells was incubated with a total of 1 μg of biotin-labeled DNA tetrahedra. The intensity of the dots in rows 2 and 3 correspond roughly with that of the 7 ng dot blot in row 1, so we estimate the total uptake of labeled DNA tetrahedra to be ~21 ng per sample. This represents approximately 2% of the total quantity of DNA tetrahedra used for transfection. Methods Supporting Figure 4 Total DNA Extraction Growth medium was removed and cells washed with PBS. 0.6ml of trypsin replacement (TrypLE Express Stable Trypsin Replacement Enzyme without Phenol Red GIBCO, USA) was then added to each sample and the samples incubated for 5 minutes at 37 o C. 1ml of PBS was then added to each sample and the resulting cell suspensions were transferred to Falcon tubes and centrifuged for 3 min at 2500 rpm. Supernatant was removed and the cell pellets resuspended in 0.4 ml extraction buffer (1M Tris-HCL ph 7.5, 5M NaCl, 500 mm EDTA, 10% SDS, 100 μg/ml RNAse A, 100 μg/ml Proteinase K). Samples were then incubated at 56 o C for 4 hours and 75 o C for 10 minutes. Cellular debris was pelleted by centrifuging at 13,000 rpm for 5 minutes. 600 μl of isopropanol was added to each sample of supernatant. Samples were mixed gently and allowed to stand for 5 minutes before being centrifuged at 13,000 rpm for 5 minutes. Pellets were washed with 1ml 70% EtOH before being air dried for 5 minutes and resuspended in 20 μl of ultrapure water. Biotin Detection Samples were applied directly to BrightStar -Plus Membranes (Ambion AM10100) and allowed to dry for approximately 1hr at room temperature. The membranes were then cross linked (15 min at 80 o C). Biotin detection was carried out using a BrightStar BioDetect Kit (Ambion AM1930) and X-Omat LS photographic film (Kodak F1149) according to the procedure described in the BrightStar BioDetect Kit manual. 6

7 DNA Complex Structures and Sequences Cy5 labeled 63-mer S4 Cy5 CCT CGC ATG ACT CAA CTG CCT GGT GAT ACG AGG ATG GGC ATG CTC TTC CCG ACG GTA TTG GAC Partial duplex of Cy5 labeled 63-mer Full duplex of Cy5 labeled 63-mer S4 Cy5 CCT CGC ATG ACT CAA CTG CCT GGT GAT ACG AGG ATG GGC ATG CTC TTC CCG ACG GTA TTG GAC S4 Partial Inverse Complement GTC CAA TAC CGT CGG GAA GAG CAT GCC CAT CCT S4 Cy5 CCT CGC ATG ACT CAA CTG CCT GGT GAT ACG AGG ATG GGC ATG CTC TTC CCG ACG GTA TTG GAC S4 Full Inverse Complement GTC CAA TAC CGT CGG GAA GAG CAT GCC CAT CCT CGT ATC ACC AGG CAG TTG AGT CAT GCG AGG Supporting Figure 5: DNA complexes used in control transfection experiments. 7

8 Cy5 labeled Tetrahedra With Lipofectin Without Lipofectin Cy5 labeled nt oligonucleotide Partial duplex of Cy5 labeled 63nt oligonucleotide Full duplex of Cy5 labeled 63nt oligonucleotide Neg. Control Supporting Figure 6: Comparison between intracellular distributions of fluorescently labeled DNA complexes used for transfection. All cell samples were analysed 24 hours after transfection. Confocal fluorescence (red) and phase contrast images are superimposed. Mean cell fluorescence readings were determined by flow cytometry. Negative control samples (Neg. Control) were transfected with unlabeled DNA tetrahedra. All other samples were transfected with DNA tetrahedra labeled with Cy5. Scale bars 100 μm. 8

9 Before Cy5 Photobleaching After Cy5 Photobleaching Supporting Figure 7: Confocal micrographs showing the photobleaching of an ROI. Cells were transfected with tetrahedra labeled with both Cy3 and Cy5. Green: Cy3 fluorescence; Red: Cy5 fluorescence; Grey: phase contrast image. Superimposed images are shown in the bottom right of each set of micrographs. The ROI is indicated by a blue line. Scale bars 50 μm. 9

10 48hr 60hr Supporting Figure 8: Intracellular localization of transfected Cy5 labeled DNA tetrahedra (additional time points for Figure 4). Images of nuclear and lysosome organelle stains are shown for reference. Time after transfection is shown in hours. Blue: nuclear stain; Red: Cy5; Green: LysoSensor (lysosomes); Grey: phase contrast. Scale bars 20 μm. 10

11 24hr Without Lipofectin With Lipofectin 48hr 60hr Supporting Figure 9: Intracellular localization of transfected DNA Cy5 labeled DNA tetrahedra (additional time points for Figure 5). Images of centrin-gfp fluorescence are shown for reference. Time after transfection is shown in hours. Green: Centrin-GFP; Red: Cy5; Grey: phase contrast. White arrows indicate the locations of pairs of microtubule organising centres. Scale bars 20 μm. 11

12 (A) (B) (C) Supporting Figure 10: Representative confocal micrographs of fixed cells transfected with fluorescently labeled DNA tetrahedra. Tetrahedra were labeled with Cy3 and Cy5. No transfection agent was used. Cells were fixed with methanol and stained with the nuclear stain DAPI. The fixed cells were scanned for fluorescence from (A) Cy3, (B) Cy5, (C) DAPI. Scale bars 50 μm. The strong nuclear localization of the transfected tetrahedra is believed to be an artefact of methanol fixation (see Supporting Note). 12