File S1 - Electroporation and Microinjection Successfully Deliver Single-Stranded and Duplex DNA into Live Cells as Detected by FRET Measurements

Figure S1: Oligo S1 HPLC analytical. Figure S2: Oligo S2 HPLC analytical. Figure S3: Oligo S3 HPLC analytical. Figure S4: Oligo S4 HPLC analytical. Figure S5: Oligo S5 HPLC analytical. Figure S6: Duplex S1:S2 in cell lysate study. No change in fluorescence is observed after S1:S2 is incubated in cell lysate at 37°C for two hours. The FRET peak at approximately 660 nm is reduced significantly after the duplex S1:S2 has been incubated with DNase for two hours. Excitation wavelength 554 nm. Figure S7: Doubly labelled single strand S3 in cell lysate study. No change in fluorescence is observed after S3 is incubated in cell lysate at 37°C for two hours. The FRET peak at approximately 660 nm disappears after S3 has been incubated with DNase for two hours. Excitation wavelength 554 nm. Figure S8: Images of single stranded Cy5 tagged DNA (S2) and single stranded Cy3 tagged DNA (S1) added to fixed/permeabilised cells respectively. Figure S9: Images of complementary Cy3 and Cy5 tagged DNA (S1 and S2) added sequentially to fixed/permeabilised cells. Figure S10: Images of Cy3 and Cy5 tagged probe DNA (S3) added to fixed/permeabilised cells. Figure S11: Images of non-complementary Cy3 and Cy5 tagged DNA (S4:S5) added together and non-complementary Cy3 and Cy5 tagged DNA (S4 and S5) added sequentially to fixed/permeabilised cells respectively. Figure S12: Mean emission spectra of regions of interest in methanol fixed cells treated with S1:S2 duplex and S3. Cells were excited with 543 nm laser only. Therefore, the peak at ca. 670 nm indicates FRET between the Cy3 and Cy5 fluorophores, hence S1:S2 and S3 are intact. Imaging was carried out using spectral imaging inverted confocal microscopy. Background regions had negligible signal. Minimum of ten cells analysed. Figure S13. Emission spectra of tagged DNA after complex formation with lipid based transfection reagent. Both S1:S2 and S3 are shown to FRET in the presence of Lipofectamine. Conditions as for transfection: 100 µM DNA, Opti-MEM medium (Life Technologies) and Lipofectamine RNAiMAX (Life Technologies). Excitation wavelength 554 nm. Figure S14: Images of Cy3 and Cy5 tagged probe DNA (S3) added to cells via lipid based transfection. Figure S15: Mean emission spectra of regions of interest in lipid based transfected cells treated with S1:S2 duplex and S3. Cells were excited with 543 nm laser only. There is no peak at ca. 670 nm which indicates a lack of FRET between the Cy3 and Cy5 fluorophores, hence S1:S2 and S3 are degraded. Imaging was carried out using spectral imaging inverted confocal microscopy. Background regions had negligible signal. Minimum of ten cells analysed. Figure S16: Images of single stranded Cy5 tagged DNA (S2) and single stranded Cy3 tagged DNA (S1) added to cells via lipid based transfection respectively. Figure S17: Images of non-complementary Cy3 and Cy5 tagged DNA (S4:S5) added together to cells via lipid based transfection. Figure S18: Images of single stranded Cy5 tagged DNA (S2) added to cells via microinjection. Figure S19: Images of single stranded Cy3 tagged DNA (S1) added to cells via microinjection. Figure S20: Images of Cy3 and Cy5 tagged probe DNA (S3) added to cells via microinjection. Figure S21: Images of non-complementary Cy3 and Cy5 tagged DNA (S4:S5) added together to cells via microinjection. Figure S22: Images of single stranded Cy3 DNA (S1) added to cells via electroporation. Figure S23: Images of single stranded Cy5 DNA (S2) added to cells via electroporation. Figure S24: Images of Cy3 and Cy5 tagged probe DNA (S3) added to cells via electroporation. Figure S25: Images of non-complementary Cy3 and Cy5 tagged DNA (S4:S5) added together to cells via electroporation. Figure S26: Cells treated with bafilomycin upon lipid based transfection of S1:S2 duplex and S3, and imaged using confocal microscopy. Images A–D are excited with the 543 nm laser only. Images E–H are excited with both the 543 and 633 nm lasers. The top row cells have been treated with the S1:S2 duplex and the bottom row cells have been treated with S3. Images of the Cy5 channel in B and D clearly show a FRET signal. Table S1: Non-complementary oligonucleotides. Table S2: HPLC retention times. Table S3: Mass spectrometry predicted and actual values. Table S4: Duplex melting temperatures. (PDF)