Biomod/2013/Aarhus/Materials And Methods/sisiRNA
Materials and methods
Click reactions of PEG and CPPs to siRNA and sisiRNA strands
The protocols for the different click reactions were established using a 5’ hexynyle modified DNA oligo. Optimizations were performed with respect to pH, incubation time and concentrations.
After establishing a general set of conditions for each of the conjugations, reactions were performed on both segments of the passenger strand of the sisiRNA construct and on the non-segmented siRNA passenger strand. The two segments of the sisiRNA strand (W179 and W004) as well as the intact siRNA strand W181 from RiboTask were bought with a 5’ hexynylamino modification and subsequently modified with a pentenoic acid.
The concentration of each strand was determined using spectrophotometry on a Nanodrop ND-1000 (Saveen Werner). Click reactions with W004, W179 and W181 to PEG1K and PEG5K were performed with 20 µM of the strands with 5 equivalents of PEG. All reactions were performed in a standard volume of 30 µL with 10 µL HEPES buffer titrated to the desired pH and 10 µL click buffer as listed in table 2. The remaining 10 µL of the total volume consisted of RNA and PEG or CPPs dissolved in dimethyl sulfoxide (DMSO), so that at least a third of the total volume was DMSO.
Reactions with PEG and melittin (H-azidopentane acid-IGAVLKVLTTGLPALISWIQQAQQL-OH, from Biosyntan) were performed at pH 7.4, as established in the optimization reactions, while reactions with GALA (H-azidopentane acid-WAALAEALAEALAEHLAEALAEALEALAA, from GL Biochem Shanghai), were made at pH 8. All reactions were incubated at 50°C on a thermo shaker for 3 hours. 2 pmol of each reaction were subsequently run on a 16 % denaturing PAGE gel and stained with SYBR Gold (Invitrogen) to estimate the yields of the reactions.
The products of the click reactions were purified using reverse-phase high performance liquid chromatography (RP-HPLC). The purified products were freeze dried and resuspended in 20 µL annealing buffer containing 200 mM potassium acetate in 20 mM HEPES (pH 7.4). The concentrations of the products were then measured using the Nanodrop, and the yield of the purifications calculated. 2 pmol of each fraction were then run on a 16 % denaturing gel to check that the correct product was eluted.
Annealing of sisiRNA
The HPLC purified, modified strands were mixed with the guide strand, W376, in annealing buffer. The samples were incubated in the PCR machine with the temperature linearly decreasing from 95°C to 4°C over 1.5 hours. Annealing reactions of all modified passenger strands to W376 as well as unmodified controls were done in a ratio of 1:1.3 with an excess of the passenger strands. To check the yield of the annealing reactions, 2.5 pmol of each annealed product were run on a 4 % aggarose gel at 4°C and stained with SYBR Gold. As a native PAGE gel needed to be run for at least 8 hours, it was tested if running an aggarose gel for between one and two hours could be used instead. A 4 % aggarose gel was compared to a 2 % gel (not shown), and it became apparent that the annealed strands could be separated well from the single strands using both percentages of the gel. However, it was observed that 2 % aggarose gel required a higher amount of sample for sufficiently intense bands to be seen, due to a higher degree of diffusion of the sample in this gel. Following this, annealing reactions were checked using 4 % aggarose gels. The yield of the annealings were estimated visually and by using the software ImageQuant TL.
Transfection of cells
KB-EGFPluc-Wagner were grown in RPMI-1640 medium containing 10 % fetal bovine serum (Gibco) and 1 % penicillin/streptomycin, L-glutamine, phenol red, without folic acid. The cells were washed with phosphate buffered saline (PBS) and released from the flask by incubating with 0.05 % Trypsin-EDTA (1X) (Gibco) for 30 minutes. The number of cells was determined using the Via 1-Casette counting chamber (Chemometer) and the computer software NucleoView NC-200. The cells were diluted in medium and seeded out as 5000 cells in 200 µL medium for each well in a 96 well plate. Four hours prior to transfection, the medium was removed, and 90 µL of fresh medium was added. Lipoplexes were formed by mixing the duplexes in different amounts with serum-free medium, Lipofectamine2000 (Invitrogen) (1 mg/mL) to a final amount of 0.3 µg for each well, followed by 30 minutes of incubation at room temperature. Cells were transfected in six or eight replicates of which half were used for cell viability (MTT) assays. A row of control wells were included as untreated controls to which only serum-free medium was added. Following all transfections, the cells were incubated for 48 hours at 37°C before measuring luciferase activity and cell viability.
Measurements of luciferase activity were performed 48 hours after transfection. The medium was removed from half of the wells, and the cells were lysed by adding 50 µL of a LAR lysis buffer containing 10 nM DTT, 125 mM TRIS base (titrated to pH 7.8 with phosphoric acid), 10 mM EDTA, 50 % glycerol and 5 % Triton X to each well. 20 µL from each of the treated wells were transferred to a white 96-well plate. A luciferase reaction buffer was prepared, containing 0.5 mM D-luciferin, 20 mM Tris-glycine buffer (pH 8), 1 mM MgClIC2, 0.25 mM CoA, 0.1 mM EDTA, 3.3 mM DTT, and 0.5 mM ATP. The reaction buffer was inserted into the FLUOstar OPTIMA plate reader which, after adding 80 µL to each well of the plate, measured the luminescence for over 10 seconds. The averages of the measured luminescence signals for each concentration of sisiRNA were normalized to the average of the untreated control and corrected for the cell viability. The values of the half maximal inhibitory concentration (IC50), were determined for each construct using GraphPad Prism 6.
Cell viability assay (MTT assay)
The MTT assay was performed on the remaining four replicates of each transfection. This was done by adding 10 µL of a solution of 5 mg/mL 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) in sterile PBS to each well and incubating for 30 minutes at 37°C until purple crystals became visible at the edges of the well. The medium was subsequently removed, and 200 µL DMSO was added to each well. The absorbance of the solutions was then measured on the FLUOstar at 590 nm and normalized to the untreated control.
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