Supporting Information for Angew. Chem. Int. Ed. Z53445 Wiley-VC 2004 69451 Weinheim, Germany
Small Interfering RNA Expression in Cells Based on an Efficiently Constructed Dumbbell-shaped DNA Masumi Taki, Yoshio Kato, Makoto Miyagishi, Yasuomi Takagi, Kazunari Taira Experimental Section: General: All solvents and reagents were commercially available and used without further purification. Assays in this study were repeated at least three times. Plasmid construction: pu6i-egfp and pu6i-lamin plasmids, encoding the U6 promoter and hairpin-type sirna expression sequence against enhanced green fluorescent protein (EGFP) and against lamin, respectively, were constructed as follows. First, we prepared sirna expression vectors using the pu6icassette vector (igene Therapeutics, Tsukuba, Japan), 10 which contains a human U6 promoter and two BspMI sites. For construction of the sirna-expression vectors, we synthesized
oligonucleotides with hairpin, terminator, and overhanging sequences. Then we annealed the fragments and inserted them into the BspMI sites of the pu6icassette vector. Sequences inserted immediately downstream of the U6 promoter were as follows : in pu6i-egfp; 5 -GGC TAT GTC TAG GAG TGT ACC TAG AAT TAC ATC AAG GGA GAT GGT GCG CTC CTG GAC GTA GCC-3, in pu6i-lamin; 5 -GGG TAA TTG GTA GAT TAA GCG GTG TGC TGT CCC GCT TGA TCT GCC AAT TGC CC-3, respectively. Conventional method to create dumbbell-shaped DNA vector using intermolecular ligation between three independent oligonucleotides; (a) PCR amplification of U6 promoter-driven sirna coding linear DNA: Five hundred pmol of each synthetic DNA primer was mixed with the vector pu6i-lamin, then a PCR reaction was carried out with Ex Taq TM DNA polymerase (TaKaRa, Shiga, Japan). The sequence of the sense and antisense universal primers for the PCR were, 5'-pGGG AAT TCA CCT GCC GGC GAG GGT TTT CCC AGT CAC GAC GTT G-3' and 5'-pGGC TGC AGA CCT GCC GGC CAC CGA GCG GAT AAC AAT TTC ACA CAG G-3', respectively. Both primers contain BspMI recognition sequence (underlined), and 5 -ends were phosphorylated. The PCR product was isolated using the Wizard (R) SV Gel and PCR
Clean-Up System (Promega, Madison, WI, USA) and digested with BspMI. After 7 hours incubation at 37 C, the digested product was isolated using Wizard (R) SV Gel and PCR Clean-Up System (Promega). (b) Conversion of the sirna-expressing linear DNA to dumbbell-shaped DNA: The uncapped PCR product was then reacted with a 10-fold excess of oligonucleotide-caps by T4 DNA ligase (DNA ligation kit; TaKaRa). Before ligation, the hairpin form of the oligonucleotide-caps presenting a sticky 5 end was obtained by boiling for 1 minute at 95 C, and subsequently cooling the sample to room temperature within 1 hour. The sequences of the oligonucleotide-caps were, 5'-pGGT GTG TCC GCG TTG GCT TTT GCC AAC GCG GAC A-3'; 5'-pCCT CGG CCT ATA GTG AGT CGT ATT AGG CGG GAA CCG CCT AAT ACG ACT CAC TAT AGG CC-3', respectively. The reaction mixture was incubated at 16 C, overnight, and the ligated product was purified by phenol/chloroform extraction, and precipitated with ethanol. Evaluation of vector stabilities in vitro by exonuclease digestion: The stabilities of linear- and dumbbell vectors were evaluated by digestion with exonuclease III (1500 U/µg of DNA, TaKaRa) at 37 C for 1 hour. An equivalent aliquot of each fraction
was electrophoresed on 8% polyacrylamide before and after the digestion reaction. After the electrophoresis, the gels were analyzed using a FluorImager 595 (Molecular Dynamics, Uppsala, Sweden), equipped with an argon laser (488 nm) as an excitation source and an emission filter (around 530 nm), to detect fluorescein s fluorescence (λ max = 492 nm). Then, ethydium bromide staining was performed with the same gels as described above. The amount of DNA and attached fluorescein was quantified by analyzing densitometry of bands using NI Image and ImageQuant programs (Molecular Dynamics), respectively. 11 Each band intensity was evaluated quantitatively from a standard curve, to quantify conversion yields from linear DNAs to dumbbell DNAs. Site-specific modification of dumbbell vector by fluorescein: For the chemical modification of dumbbell vectors, we used the Fluorescein Amine Labeling Kit (Panvera, Madison, WI) according to the instruction manual. Briefly, a 67 nm dumbbell vector in 30 µl of 100 mm phosphate buffer (p 7.0) solution containing 4 mm succinimide ester of fluorescein was incubated at 37 C for 1 hour. After incubation, the reaction was stopped by adding 100 mm Tris/Cl buffer (p 8.0) and the resulting
solution then incubated at room temperature for an additional 30 minutes. DNA was isolated using the Wizard (R) SV Gel and PCR Clean-Up System (Promega). Cells, cell culture, transfection, and GFP fluorescence assay: ela S3/EGFP cells were generated as follows. ela/s3 cells were transfected with linearized pygegfp (Clontech, East Meadow Circle, PA), and hygromycin-resistant clones were selected in Dulbeco's modified Eagle's medium (DMEM; Sigma, St. Louis, M) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; GIBC-BRL, Gaithersburg, MD), hygromycin (100 µg/ml; Sigma) and an antibiotic-antimycotic mixture (GIBC-BRL). ne of the clones, ela S3/EGFP clone 3 was used for the experiments in this report, and was maintained in the above medium at 37 C, 5% C 2 in a moist atmosphere. These cells were grown to approximately 40-50% confluence in each 8-well chambered glass slide (Nalge Nunc International, Naperville, IL). Then, transfections of the DNAs were carried out using PTI-MEM medium (Invitrogen, Carlsbad, CA) and Trans-It LT-1 reagent (PanVera, Madison, WI) according to the manufacturer's protocol. They were independently transfected with 100 ng of dumbbell-shaped vector, linear PCR product vector, or pu6i-egfp plasmid vector encoding sirna targeted against the EGFP gene. As a negative control, they were
also transfected with 100 ng of dumbbell-shaped vector targeted against the lamin gene. Mock transfection was carried out without using any DNA. Incubation was performed for 48 hours. At 24 hours post-induction, the medium was replaced by fresh growth medium containing hygromycin and the antibiotic-antimycotic mixture. Living cells on the glass coverslips were observed after 48 hours using a conventional fluorescence microscope (LSM-510, Carl Zeiss, berkochen, Germany). Inhibition of nuclear envelope proteins (lamin A/C): ela S3 cells were transfected with 100 ng of dumbbell-shaped vector encoding sirna targeted against lamin gene, according to the above procedure. The transfected cells grown on glass coverslips were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 minutes at room temperature. The slides were rinsed twice in PBS and drained. The cells were immersed in PBS buffer with 0.1% Triton X-100, and incubated for 30 minutes. The slides were rinsed twice in PBS and drained. The cells were blocked with 1% BSA and 0.1% Triton X-100 in PBS and then incubated with a 1:1000 dilution of monoclonal 636 lamin A/C specific antibody (Santa Cruz Biotechnology) for 1 hour at room temperature. The antibody was drained, and the slides were rinsed in the BSA/Triton X-100/PBS solution and then incubated with a 1:1000 dilution of
Alexa-568-conjugated anti-mouse IgG (A11031; Molecular Probes) for 1 hour at room temperature. The antibody was drained and slides were rinsed. The coverslips were inverted and mounted with Prolong antifade (Molecular Probes). The cells on the coverslips were observed using a conventional fluorescence microscope (Zeiss).
A: Dumbbell vector targeted against lamin B: No vector Phase contrast lamin A/C Superposed Figure S1. Inhibition of nuclear envelope proteins (lamin A/C) expression using dumbbell vector in ela S3 cells.
R N N ptnaggagtt P TCTCCTNAGC... - R 1a : C 3 1b : N N 2 1c : N N N C Figure S2. Detailed structures of the sense primers 1a-c.
e.g. sirna coding region A B 1. PCR A promoter gene to be transcribed B cdna A 2. Digestion using restriction enzyme(s). 3. Purification B A B 4. Intermolecular ligation airpin DNA in the loop region 5. Purification Scheme S1. Previously published conventional dumbbell-cloning method. 6,9
Scheme S1. Previously published conventional dumbbell-cloning method. 6,9 Intramolecular ligation between three different molecules lowers the conversion yield from linear DNA to dumbbell DNA. Excess amounts of hairpin DNAs in the loop region must be required, and almost all hairpin DNA molecules remain unreacted.