Computational protocol: Carrier-free Gene Silencing by Amphiphilic Nucleic Acid Conjugates in Differentiated Intestinal Cells

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[…] Materials. HCT-116 cell line was kindly provided by Prof. Azzalin's group at ETH Zurich and Caco-2 cells were purchased from ATCC (Manassas, VA). Dulbecco's modified essential medium (DMEM) with GlutaMAX, Opti-MEM medium, fetal bovine serum (FBS), nonessential amino acids, penicillin-streptomycin solution, trypsin, Lipofectamine 2000 (LF), phosphate-buffered saline (PBS; 1 mmol/l KH2PO4, 3 mmol/l Na2HPO4, 155 mmol/l NaCl, pH 7.4), and RNase-free distilled water were obtained from Invitrogen (Carlsbad, CA). Porcine pancreatin (4×USP), ammonium persulfate, triethylammonium acetate (TEAA) buffer 1 mol/l, Intralipid (20% w/v of soybean oil, 1.2% of egg yolk phospholipids, 2.25% of glycerol, pH 6–8.9; mimic of high-fat meal), chloroform (CHCl3), Triton X-100, NaF, Na3VO4, Tris, and skim milk powder were obtained from Sigma-Aldrich (Buchs, Switzerland). Sodium decanoate was purchased from TCI (Tokyo, Japan). Duplex annealing buffer (100 mmol/l potassium acetate, 30 mmol/l HEPES, pH 7.5) was purchased from Integrated DNA Technologies (IDT, Coralville, IA). Potassium dihydrogen phosphate was obtained from Merck (Kenilworth, NJ). Methanol (MeOH), N,N,N′,N′-tetramethylethylenediamine (TEMED), and phenylmethylsulfonyl fluoride (PMSF) were purchased from Acros Organics (Geel, Belgium). Hexafluoroisopropanol (HFIP) was obtained from Fluorochem (Hadfield, UK). Ethylenediaminetetraacetic acid (EDTA) and polysorbate 20 were purchased from AppliChem (Darmstadt, Germany). Gel Red nucleic acid gel stain was obtained from Biotium (Hayward, CA). MycoAlert PLUS Mycoplasma Detection Kit was purchased from Lonza (Basel, Switzerland). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies (Rockville, MD). Thermanox coverslips, DNA loading dye, NaCl, and micro BCA protein assay kit were purchased from Thermo Fisher Scientific (Waltham, MA). Transwell inserts were obtained from Corning (Corning, NY). Low-binding microcentrifugation tubes (DNA Lobind) were purchased from Eppendorf-Vaudaux (Schönenbuch, Switzerland). RNeasy Mini kit and specific primers for α-splicing variant of human Bcl-2 mRNA (Hs_BCL2_1_SG; QT00025011) and human β-actin (Hs_ACTB_2_SG; QT01680476) were obtained from Qiagen (Valencia, CA). High-capacity cDNA reverse transcription kit and Power SYBR Green polymerase chain reaction (PCR) Master Mix were purchased from Applied Biosystems (Foster City, CA). Complete EDTA-free protease inhibitors' cocktail was purchased from Roche Diagnostics (Mannheim, Germany). Poly(vinylidene difluoride) membranes (PVDF) were obtained from Bio-Rad Laboratories (Hercules, CA). Mouse antihuman Bcl-2 monoclonal antibody and horseradish peroxidase (HRP)-conjugated goat antimouse IgG polyclonal antibody were purchased from Dako (Glostrup, Denmark). Rabbit anti-β-actin polyclonal antibody and HRP-conjugated goat antirabbit IgG polyclonal antibody were obtained from Abcam (Cambridge, UK). ImmunoCruz Western blotting luminol reagent was purchased from Santa Cruz Biotechnology (Dallas, TX). Super RX X-Ray films were obtained from Fujifilm (Tokyo, Japan).Synthesis of oligonucleotides and their derivatives. Unmodified Bcl-2 targeting siRNA and negative control siRNAnc with nontargeting sequence were synthesized by Bioneer (Daejeon, South Korea). Unmodified antisense strand of siRNA was obtained from Microsynth (Balgach, Switzerland). All ONs were synthesized according to the standard protocol for automated phosphoramidite solid-phase synthesis except for L-OMe conjugate, which was provided by Microsynth. DSA was conjugated via an aminohexanol-linker to the 5′-end of ONs in line with previously described method. All L-ON conjugates were purified by reverse-phase HPLC, analyzed by LC-MS, and quantified via UV spectrophotometry (NanoPhotometer P 330, Implen, Germany). Details of synthetic procedure and analytical data are presented in the supporting information. The ONs' molar extinction coefficients at 260  nm were calculated using the software of IDT website (OligoAnalyzer tool, FANA and MOE extinction coefficients were calculated using DNA and OMe values, respectively. These modifications were assumed to have negligible effect on the extinction coefficients, as previously described., The complementary single strands of siRNAs were combined in duplex annealing buffer at a concentration of 50 µmol/l each, heated up to 95°C for 1 minute, and cooled slowly to 4°C overnight to ensure proper annealing. Various ONs and L-ON conjugates were dissolved in RNase-free deionized water at a concentration of 100 µmol/l and stored at −20°C.Cell culture. HCT-116 cells were maintained in DMEM medium supplemented with GlutaMAX containing 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37°C in a 5%-CO2 humidified atmosphere. The cells were seeded in a 24-multiwell plate at a density of 4 × 104 cells/well. For the inverted transfection, the cells were seeded on Thermanox coverslips in a 24-multiwell plate at a density of 1 × 105 cells/well. The cells were incubated for 1 day before experiments. Caco-2 cells were maintained in DMEM medium supplemented with GlutaMAX containing 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 1% of nonessential amino acids at 37°C in a 5%-CO2 humidified atmosphere. Cells with passage number between 58 and 77 were seeded in a 12-multiwell plate at a density of 7 × 104 cells/well. For the differentiated Caco-2 cell monolayer transfection, the cells were seeded in Transwell inserts with polyester membrane with pore size of 0.4 µm in a 12-multiwell plate at a density of 1.12 × 105 cells/well as previously described. The medium was exchanged every other day, and cells between 13 and 17 days of differentiation were used for the experiments. The differentiation of monolayers was monitored by measuring TEER using an EVOM epithelial voltmeter with STX2 electrode (World Precision Instruments, Sarasota, FL).All the monolayers achieved TEER higher than 1,000 Ωcm after the 2 weeks of culturing, indicating the completion of differentiation process. TEER values of individual wells measured just before the transfection with ONs were set to 100% and their change was monitored for the three following days.All experiments were performed on mycoplasma-free cell lines (regularly checked by MycoAlert PLUS Mycoplasma Detection Kit), and only cells in the exponential phase of growth were used for seeding.Cytotoxicity assay. To compare the cytotoxicities of different L-ON conjugates, HCT-116 cells were seeded in a 96-multiwell plate at a density of 7 × 103 cells/well the day before the experiment. Alternatively, Caco-2 cells were seeded in a 96-multiwell plate, and the culture medium was exchanged every other day for 2 weeks to obtain differentiated monolayers. The cells were treated with various concentrations of L-ON conjugates in 50 μl of serum-deficient Opti-MEM medium for 15 hours. The Opti-MEM medium containing no L-ON was used as a control. The medium was exchanged for 100 μl of DMEM supplemented with 10% FBS, and the cells were further incubated for 24 hours, after which the cell viability was assessed using tetrazolium-based CCK-8 reagent following the manufacturer's instructions.Screening of Bcl-2 mRNA silencing efficiencies of various L-ONs. To assess the silencing efficiency of various L-ON conjugates, the conjugates at various concentrations (0.25 – 1 µmol/l) were incubated with HCT-116 and Caco-2 cells. Given the absence of intact serum in the intestinal environment, all transfection experiments were performed in serum deficient Opti-MEM medium. The Opti-MEM medium containing no ONs was used as a control. Following overnight incubation (15 hours), the transfection medium was exchanged with fresh DMEM supplemented with 10% FBS. After 2.5 days of further incubation cells were washed with PBS, and total RNA was isolated using RNeasy Mini kit (Abs260 /Abs230 >1.8) according to the previously optimized method. The expression levels of Bcl-2 mRNA relative to the internal control β-actin mRNA were quantified by two-step quantitative real-time PCR. Briefly, cDNA was synthesized from 1.2 µg of total mRNA using high-capacity cDNA reverse transcription kit according to the manufacturer's instructions. Quantitative real-time PCR was performed using Power SYBR Green PCR Master Mix and specific primers for human Bcl-2 and β-actin on a 7900HT Fast Real Time PCR instrument (Applied Biosystems) according to the manufacturer's instructions. Briefly, the reaction mixtures were incubated at 50°C for 2 minutes and 95°C for 10 minutes followed by 40 cycles of denaturation (95°C for 15 seconds) and extension/detection (60°C for 1 minute). Relative mRNA expression levels were calculated using the delta delta Ct (2-ΔΔCt) method (the fluorescence threshold was set to 0.4). Results are expressed as the Bcl-2 mRNA level change between ON-treated and ON-free medium treated cells. L-ON potency (effective concentration causing 50% of target mRNA silencing, EC50) was calculated using a four-parameter logistic function to fit the dose-response data via SigmaPlot 13.0 software.Comparison of transfection efficiencies in upright and inverted transfection. The transfection medium consisted of 250 μl of Opti-MEM containing either 1 μmol/l of carrier-free Bcl-2 targeting L-FANA conjugate or 0.05 μmol/l of Bcl-2 specific siRNA complexed with LF according to the manufacturer's instructions as a particle-mediated delivery control. HCT-116 cells grown on the coverslips were washed with Opti-MEM, and the coverslips were transferred using tweezers into a new 24-multiwell plate for the transfection. For the upright transfection, the cells were placed on the bottom of the multiwell plate followed by the addition of the transfection medium. For the inverted transfection, the transfection medium was added to the empty well, and the cells grown on coverslips were carefully deposited upside down onto the surface of the medium. The coverslips floated on the surface of the medium due to the surface tension of the medium. After overnight incubation (15 hours), the cells were transferred to a new plate and further cultured for 2.5 days in 500 μl of DMEM supplemented with 10% FBS. Subsequently, total RNA was isolated and gene expression levels of Bcl-2 mRNA were assessed as described above.L-FANA conjugates stability in SIF and 0.5% soybean oil emulsion. To investigate the influence of digestive enzymes and food-derived fats present in intestine on the biological function of L-FANA, its stability and efficacy were tested in simulated intestinal environment containing pancreatic enzymes and lipids. For polyacrylamide gel electrophoresis analysis, 1 μl of L-FANA (100 μmol/l) was mixed with 9 μl of USP SIF (2.5 g/l porcine pancreatin (4 × USP), 50 mmol/l KH2PO4, pH 6.8) or water and incubated for 2 hours or 15 hours at 37°C. Samples were kept at 95°C for 15 minutes to heat-inactivate the enzymes and mixed with 1 µl of DNA loading dye. Alternatively, 1 μl of L-FANA was premixed with 1 μl of 5% soybean oil emulsion in Opti-MEM (prepared by diluting 20% Intralipid with Opti-MEM) for 30 minutes followed by incubation at 37°C for 2 hours with 8 μl of active/heat-inactivated SIF or water. Samples were then loaded onto 20% (w/v) polyacrylamide gel prepared in a Tris-acetate-EDTA buffer (TAE: 40 mmol/l Tris-acetate, 1 mmol/l EDTA, pH 8.0) by free-radical polymerization with ammonium persulfate/TEMED as an initiator. The gel was then immersed in TAE buffer and electrophoresed at constant voltage of 150 V for 60 min. L-FANA was revealed following manufacture's protocol for Gel Red nucleic acid gel stain, and fluorescence was recorded on a ChemiDoc XRS (Bio-Rad Laboratories).For the LC-MS analysis, 2 μl of L-FANA (100 μmol/l) were mixed with 18 μl of SIF or water and incubated for 2 hours at 37°C, then at 95°C for 15 minutes, and centrifuged at 14000 × g at room temperature for 10 minutes. The supernatants were mixed with 50 μl of CHCl3, and shaken to extract possible hydrophobic contaminants from SIF. After incubating the mixtures at room temperature for a few minutes, water layers were collected and stored at −20°C until analysis. Analytical liquid chromatography-mass spectrometry (LC-MS) was carried out on Agilent LC-MS using a reverse phase column (Waters Acquity OST C18, 2.1 x 50 mm, 1.7 µm) with solvent A being 400 mmol/l HFIP and 15 mmol/l TEAA in water and solvent B being MeOH. The flow was set at 0.3 ml/min, and a gradient was run at 65°C from 5 to 90% B in 14 minutes.Transfection of L-FANA preincubated with SIF and 0.5% soybean oil emulsion. The influence of preincubation of L-FANA conjugates with SIF on the transfection efficacy was investigated. HCT-116 cells were seeded one day prior to the experiment in a 24-multiwell plate at a density of 4 × 104 cells/well in DMEM containing 10% FBS. Two and a half microliters of L-FANA were pre-incubated with 22.5 µl of SIF for 2 hours at 37°C followed by heat-inactivation of enzymes for 15 minutes at 95°C. The mixtures were diluted ten times with Opti-MEM to a final L-FANA concentration of 1 μmol/l and incubated with cells overnight (15 hours). ON-free Opti-MEM medium was used as a control. Subsequently, the transfection medium was changed to DMEM supplemented with 10% FBS. After 2.5 days of further incubation, total RNA was isolated, and gene expression levels of Bcl-2 mRNA were assessed as described above. To study the silencing activity of L-FANA released from SIF-digested oil emulsion, 2.5 μl of L-FANA (100 μmol/l) were preincubated with 6.25 µl of 20% Intralipid for 30 minutes at room temperature followed by incubation with 25 µl of SIF for 2 hours at 37°C. The released L-FANA was separated from cytotoxic oil digestion products by preparative polyacrylamide gel electrophoresis before transfection experiments. Gels were prepared and electrophoresed as described above. After the electrophoresis, the L-FANA band was excised, weighed, and immersed in 250 μl of Opti-MEM. For L-FANA extraction, samples containing gel pieces were flash-frozen in liquid nitrogen, heated at 90°C for 15 minutes, and agitated at room temperature for 3 hours. The concentration of extracted L-FANA in the supernatant was 0.6 µmol/l. The supernatants were incubated with cells overnight (15 hours). The expression levels of Bcl-2 mRNA were assessed as described above.Transfection of differentiated Caco-2 cell monolayers. The differentiated Caco-2 cell monolayers grown in Transwell inserts for 2 weeks were washed with Opti-MEM medium from apical and basal sides. The 500 µl of Opti-MEM medium containing either naked ON derivative (0.625 – 5 µmol/l) or siRNA (0.2 – 0.4 µmol/l) complexed with LF according to the manufacturer's instructions was added to the apical compartment. The basal chamber was filled with 1.5 ml of Opti-MEM medium. The Opti-MEM medium in both compartments was used as a control. Following the overnight incubation (15 hours), the transfection medium was exchanged with fresh DMEM supplemented with 10% FBS in both compartments. For Bcl-2 mRNA expression analysis by qRT-PCR, cells were lysed 2.5 days after transfection as described above. For Bcl-2 protein expression analysis by western blot, cell monolayers were lysed 4 days after transfection in 25 µl of lysis buffer (20 mmol/l Tris–HCl pH 7.7, 150 mmol/l NaCl, 5 mmol/l EDTA, 1% v/v Triton X-100, 25 mmol/l NaF, 1 mmol/l PMSF, 1 mmol/l Na3VO4 supplemented with Complete protease inhibitors) in line with previously described method. Briefly, cell lysates were scraped from the Transwells, centrifuged at 10,000 × g for 15 minutes at 4°C to remove cell debris, and protein concentration in the supernatants was determined by the micro BCA assay according to the manufacturer's instructions. 50 µg of total protein per sample were resolved on 12% SDS-PAGE under reducing conditions and transferred to a PVDF membrane. The membrane was washed once with TBS-T buffer (20 mmol/l Tris–HCl pH 7.7, 150 mmol/l NaCl, 0.1% v/v polysorbate 20) and blocked with TBS-T containing 5% w/v skim milk (blocking buffer) for 1 hour. The membrane was cut in two at 35 kDa; the lower part containing Bcl-2 (26 kDa) was incubated with anti-Bcl-2 antibody diluted to 1:100 in blocking buffer, and the upper part containing β-actin (42 kDa, loading control) was incubated with anti-β-actin antibody diluted to 1:4,000 overnight at 4°C. Membranes were washed 3 times for 5 minutes with PBS-T followed by 1.5-hour-incubation with the corresponding HRP-conjugated secondary antibodies diluted to 1:4,000 in blocking buffer. Membranes were washed three times with TBS-T, and protein bands were detected with ImmunoCruz luminol reagent and revealed on Super RX X-Ray films using an AGFA Curix 60 film processor (AGFA, Mortsel, Belgium). The relative intensities of the bands were analyzed using Image J software (National Institutes of Health, Bethesda, MD).Statistical analysis. All treatment groups were compared pairwise using the one-way analysis of variance (ANOVA) test combined with Tukey's (Holm-Sidak) post-hoc test assuming normal data distribution. The statistical analysis was performed using SigmaPlot 13.0 software. The differences between treatment groups were considered statistically significant at P-values <0.05. Figure S1. Viability of HCT-116 cells after transfection with various ON derivatives at a dose of 1 µmol/l in Opti-MEM medium overnight. Cell viability was assessed 1 or 2.5 days after medium exchange. Figure S2. Bcl-2 mRNA silencing and viability of HCT-116 cells after transfection with various siRNA derivatives complexed with LF and free PS siRNA. Figure S3. The Bcl-2 mRNA silencing kinetics in HCT-116 cells transfected with 1 µmol/l of L-FANA or 0.05 µmol/l of siRNA/LF in Opti-MEM medium. Figure S4. The influence of incubation time on knockdown efficacy in HCT-116 cells transfected with L-FANA or siRNA/LF lipoplex. Figure S5. Polyacrylamide gel electrophoresis images of L-FANA conjugate and unconjugated FANA incubated with oil emulsion. Figure S6. Transfection of proliferating Caco-2 cells with siRNA/LF and negative control siRNA (siRNAnc)/LF lipoplexes. […]

Pipeline specifications

Software tools OligoAnalyzer, SigmaPlot
Applications Miscellaneous, qPCR
Diseases Gastrointestinal Diseases, Intestinal Diseases, Inflammatory Bowel Diseases, Drug-Related Side Effects and Adverse Reactions