Sequencing and transcription of the scaffold
To be able to sequence the entire scaffold the template DNA was first amplified with PCR and then cloned into a pUC18 vector. The transcription of scaffold was done using a PCR product of the template DNA.
Amplification of DNA template
PCR was used to amplify the DNA sequence used as template in the transcription of the RNA scaffold. The template for the PCR was a plasmid used for Luciferaseassays, kindly provided by Jesper B. Bramsen. A mixture with a final volume of 100 μl was made containing 1X Taq Reaction buffer (200mM Tris pH 8.4, 500mM KCl), 2.5mM MgCl2, 200μM dNTP mix, 200nM forward primer (FW-Luc-T7-EcoRI), 200nM backward primer (BW-Luc-BamHI), 1μM Taq DNA polymerase, 1 μl template (genomic siCheck2 siEGFP1 with an unknown concentration). The concentration of the template was unknown. Primer sequences are listed in Table A.2. The buffer and polymerase was from Invitrogen. The PCR machine used was a VWR Collection DOPPIO Thermal Cycler. The program was 27 cycles of; 94°C for 20 seconds, 55°C for 20 seconds and 72°C for 20 seconds. The ramp time was 3°C/s and the PCR machine stored the mixture at 10°C. The PCR product was purified using a illustra GFXTM PCR DNA and Gel Band Purification Kit from GE Healthcare and following the provided procedure. 500 μl Capture buffer type 3 was added to the 100 μl sample and vortexed. The mixture was added to GFX MicroSpinTM column, spun for 30 seconds at 16.000 rcf and the flow through was discarded. 500 μl Wash buffer type 1 was added to the column, spun for 30 seconds at 16.000 rcf and the collection tube was discarded. 50 μl Elution buffer type 6 (nuclease-free water) was added to the column, which was inserted into a clean eppendorf tube. After one minute the column was spun for one minute at 16.000 rcf, eluding the template into the eppendorf tube.
Digestion and ligation of plasmid and PCR product
The purified PCR product was digested with the restriction enzymes EcoRI and BamHI. Fermentas FastDigest R enzymes were used and the procedure provided by the manufacturer was followed. A mixture with a total volume of 35 μl containing the purified PCR product, 1X FastDigest R buffer, 1X EcoRI, 1X BamHI was incubated at 37°C for 30 minutes. Another mixture with a total volume of 20 μl containing 1 μg pUC18, 1X FastDigest R buffer, 1X EcoRI, 1X BamHI was incubated at 37°C for 30 minutes. The digested PCR product and plasmid was purified following the procedure explained earlier for the GFXTM Purification Kit.
The enzyme and buffer used for the ligation were from Fermantas. The reaction was carried out in a total volume of 20 μl by mixing 250 ng digested pUC18, ~4- fold excess of digested PCR product, 1X T4 DNA Ligase buffer, 1X T4 DNA Ligase (200 CEU/μl) and incubating at 17°C over night.
Transfection of cells
Prepared heat-shock cells (E. coli XL1-blue from Stratagen) was transfected by adding 5 μl of the ligation product to 200 μl cells and incubated on ice for 40 min. The cells were heat-shocked at 42°C for 100 seconds and incubated on ice for 5 min. After the addition of 800 μl of LB media the mixture was incubated at 37°C for an hour. The cells were then spun down for 30 sec. at 12,000 rpm and ~900 μl of the supernatant was removed. After resuspension the cells were diluted 1.000x and plated on agar and incubate at 37°C over night. Cells were then replated and incubated at 37°C over night.
The agar plates were made by mixing 10 g Typtone, 5 g yeast extract, 10 g NaCl, 15 g Agar and adding double distilled water to a final volume of one liter. The mixture was autoclaved and cooled to 55°C and X-gal and Ampicillin were added to a final concentration of 20 μg/ml and 50 mg/ml, respectively.
Cells were transfered to a tube that contained 5 ml LB media and Ampicillin (100 mg/ml), using a autoclaved toothpick. The solution was incubated at 37°C over night while shaking at 200 rpm. The cells where then spun down for 2 min. at 7.000 rcf and the media was removed.
Purification of ligation product
GeneJETTM Plasmid Miniprep Kit from Fermentas was used to purify the ligation product from the cells. The procedure provided by Fermentas was as followed. The pelleted cells were resuspended in 250 μl Resuspension Solution (containing RNase A) by vortexing. The solution was mixed with 250 μl Lysis Solution by inversion, followed by the addition of 350 μl Neutralization Solution and then centrifuged for 5 min at 13.000 rcf. The supernatant was loaded on the GeneJETTM spin column and centrifuged for 1 min. at 13.000 rcf. The column was washed two times by adding 500 μl Wash Solution, centrifuging for 1 min. at 13.000 rcf and discarding the flow-through. After the second wash the column was centrifuged for 1 min. more at 13.000 rcf. The coulmn was transfered to a clean eppendorf tube, 50 μl Elution Buffer was added and after 2 min. the column was spun for 2 min. at 13.000 rcf, eluding the purified ligation product.
BigDye® Terminator v3.1 Cycle Sequencing Kit was used and the procedure provided by the manufacturer was followed. Two reactions were performed, one in each direction, to ensure higher fidelity in the analysis of the sequencing. The sequencing mixture contained 200 ng plasmid DNA, 5 pmol primer (only forward or backward), 1X BigDye Sequencing Buffer, 1X BigDye Ready Reaction Premix. The primers used were standard sequencing primers ’FW M13 (-20)’ and ’BW M13’ (see the sequences in Table A.2). Same machine and program as for PCR was used for the sequencing reaction. After the reaction water was added to 20 μl and the mixture was delivered to Hans Hjorth who purified and sequenced it. CLC Main Workbench was used to analyze the sequences.
In vitro transcription
The template for the transcription of the scaffold was a PCR product with a T7 promoter incorporated upstream of the scaffold sequence. The PCR was carried out as the amplification procedure explained earlier, but the primers used were ’FW-Luc-T7-EcoRI’ and ’BW-Luc’. See sequences in Table A.2.
MEGAscript™ High Yield Transcription Kit from Ambion was used and the manufacturers procedure was followed. A mixture with a total volume of 20 μl was made, containing 1X MEGAscript Enzyme Mix (T7), 1X Reaction Buffer, 7.5mM NTP mix (T7) and template with an expected concentration of ~1 μM. The content was mixed thoroughly and incubated at 37°C over night. The template was removed by adding 1X TURBO DNase (2 U/μl) and incubating at 37°C for 15 min.
Purification of RNA scaffold
A denaturing 6% polyacrylamide gel (PAG) was made, with SequaGelTM Urea- Gel System from National Diagnostics by mixing 1X TBE buffer, 1X Concentrate, 1X Diluent, 2.5μM Tetramethylethylenediamine (TEMED), 7μM Ammonium persulfate (APS) in a total volume of 40 ml and casting the gel between two glass plates. The gel was pre-run for a total of 25 min. at 15Wand the wells were cleaned, before loading the transcription product. The gel was run for 45 min. at 15 W and the band of interest was cut out using a scalpel. The cut-out was transfered to an elution buffer containing 330 μl TE buffer with 200mM NaOAc and 100 μl phenol (pH 6.6). The elution mixture was set to shake at 800 rpm over night. After elution 200 μl phenol was added to the mixture, which was vortexed for 30 sec. and spun at 14.000 rcf for 2 min. The following three step were all vortexed and spun in the same way. 1) The supernatant was transfered to a new effendorf tube containing 300 μl phenol. 2) The supernatant was transfered to a new effendorf tube containing 150 μl phenol and 150 μl chloroform. 3) The supernatant was transfered to a new effendorf tube containing 300 μl chloroform. The RNA was then presipitated by transferring the supernatant to a new effendorf tube containing 750 μl 96% ethanol. The mixture was vortexed for 30 sec. and then put on dry ice for 15 min. After centrifuging at 4°C for 15 min. at 14.000 rcf, the supernatant was discarded and 450 μl 70% ethanol was added. After centrifuging again at 4°C for 15 min. at 14.000 rcf, the supernatant was removed completely and the pellet was resuspended in 20 μl RNase-free water. The concentration was measured by OD260.
Quality control of staple strands
The staples were run on a gel with the scaffold to determine weather or not they were contaminated with RNase. The FRET staples were further tested by measuring the absorption and thereby determining the concentration of fluorophores compare to the concentration of DNA.
A 1.5% agarose gel was made by mixing 0.9 g UltraPureTM Agrose from Invitrogen and 0.5X TBE in a total volume of 60 ml. The solution was heated in the microwave oven until the agarose was dissolved. After the solution had cooled, 1X SYBR® Safe was added and the solution was poured into the mold and cast using a comb with twelve teeth. The gel was electrophoresed for 20 minutes at 120 V in 0.5X TBE. ’Native’ agarose gels were also used, with the only difference being the addition of 5 mM MgAc to the gel and buffer as well as a change in the electrophoresis conditions (2 hours in 5°C at 100 V). A Typhoon TRIO+TM Variable Mode Imager was used to visualize the gels. The excitation was at 532 nm and the emission filter was 580 BP 30.
Absorption measurements were used to determine the quality of the FRET oligos, by calculating the amount of DNA in a sample compared with the amount of fluorophores based on the absorbance. A 2μM solution of each of the four FRET oligos (sequences listed in Table A.2) were made using nuclease-free water as solvent. 65 μl was pipetted to a quartz cuvette with an optical path length of 3mm. A cuvette with nuclease-free water was used as reference. The spectrophotometer used was a Shimadzu UV-3600 UV-VIS-NIR and the spectra were recorded from 200nm to 800 nm.
The S structure was self-assembled by the following procedure. The staples were first treated with SDS by mixing 10 μM of each staple strand, 1% SDS and 63 mM Tris pH 7.5 in a total volume of 50 μl. The mixture was heated to 95°C for 2 minutes. Then scaffold, TAE buffer and MgAc was added so the final concentrations were 0.686 μM scaffold, 1X TAE, 12.5 mM MgAc with a total volume of 70 μl.
The F structures were self-assembled by the following procedure. The staples were first treated with SDS by mixing 8.33 μM of each staple strand, 1% SDS and 63 mM Tris pH 7.5 in a total volume of 60 μl. The mixture was heated to 95°C for 2 minutes. Then scaffold, TAE buffer and MgAc was added so the final concentrations were 0.6 μM scaffold, 1X TAE, 12.5 mM MgAc with a total volume of 70 μl. Staple strand composition of the different structures are shown in Table 2.1.
A PCR machine (VWR Collection DOPPIO Thermal Cycler) was used to perform the annealing. The program was 95°C for 5 minutes, a ramp of 3°C/s to 80°C, -0.5°C/cycle to 60°C with a cycle-time of 10 seconds, -0.5°C/cycle to 40°C with a cycle-time of 60 seconds and -0.5°C/cycle to 20°C with a cycle-time of 90 seconds. The PCR machine stored the mixture at 10°C.
Purification of the structure
In order to use the structures for fluorescence studies they had to be purified, which was done by HPLC and subsequently concentrated using a filter-column.
The self-assembled structure was purified by HPLC using a BioSepTM-SEC-s2000 column from Phenomenex. It is a silica-based resin column with a particle size of 5μm and a pore size of a 145 Å.1 The HPLC apparatus was a Agilent 1100 Series HPLC system. The buffer was set at pH 7.5 and contained 20 mM Tris, 100 mM KaCl, 10 mM MgCl, with a flow rate of 1 ml/min. A total volume of 60 μl of the assembled structure was loaded in the HPLC. Fraction detection with Agilent Diode Array Detector UV/VIS was done at 260 nm (DNA), 560 nm (Cy3) and 650 nm (Cy5). The program used for interpreting data was Agilent ChemStation for LC 3D Systems. Elution data can be found in Figure B.3 and B.4.
F1 was used to calibrate, so 24 fractions were collected from 4 min. to 7 min., with ~125 μl in each well. The fractions used was the 6 fractions (~750 μl) from 6:15 to 7:00. For F2-8 24 fractions were collected from 5 min. to 9 min., with ~165 μl in each well. The fractions used was the 11 fractions (~1.8 ml) from 6:10 to 8:00.
Concentration of the purified samples were done using Amicon® Ultra-0.5 Centrifugal Filter Devices from Millipore. The filter-columns had a Nominal Molecular Weight Limit (NMWL) of 100,000. Up to 500 μl of the sample was added to the filter-column and spun at 14.000 rcf for 5 min. The procedure was carried out four times, adding all of the purified sample to the column. After the last addition the sample was spun for 10 min. To collect the concentrated sample, the column was turned upside down into a clean eppendorf tube and spun at 1.000 rcf for 3 min. The samples were then diluted to a total volume of 65 μl for the FRET measurements.
FRET measurements and normalization
A FluoroMax-3 spectrofluorometer from Horiba Scientific was used for the fluorescence measurements. Three different setting were used to measure FRET. Setting 1 excited the sample at 530 nm and measured the spectrum from 540 nm to 750 nm. This setting excited the donor and measured the emission spectrum of both donor and acceptor. Setting 2 excited the sample at 600 nm and measured the spectrum from 610 nm to 750 nm. This setting excited the acceptor and only measured the emission spectrum of the acceptor. Setting 3 was a time-resolved measurement that excited the sample at 530 nm every 30 second for 600 seconds, and measured the fluorescence at 560 nm (Cy3 donor) and 665 nm (Cy5 acceptor). The shutter was closed between measurements to eliminate photobleaching. This setting excited the donor and measured the emission maxima of both donor and acceptor. All measurements were done by pipetting 65 μl sample into a quartz cuvette, which had an optical path length of 3 mm.
The spectra of F1, F2 and F4 was normalized to that of F3 and the emission spectrum of the S structure was used as ’background’. The normalization procedure is shown in Figure 4.1, with the F2 structure as an example. First the background was deducted from the emission spectra of the four structures. The fluorescence intensity of the direct excitation of Cy5 (setting 2) only differs with concentration between the four structures and was therefore chosen as normalization parameter. The fluorescence intensities were compared at 666 nm and F3 had the highest intensity. The normalization wavelength was chosen based on the Cy5 maxima at 667 nm, 664 nm, 668 nm and 665 nm for F1, F2, F3 and F4, respectively. The normalization factors found were applied to the spectra of F1, F2 and F4.
The SAXS measurements were performed at the SAXS laboratory at the Chemical Department at University of Aarhus on an optimized version of Bruker Nanostar SAXS machine. 60 µl DNA/RNA sample is loaded into a quarts capillary. The sample is placed in the vacuum chamber of the SAXS machine. The beam is attenuated and the direct intensity through the sample is recorded over a period of 10 seconds. This measurement is made to control the instrument settings. The SAXS data is recorded over 5 periods of 300 seconds. The splitting into 5 periods is to avoid loss of all data if the sample moves during recording. The scattering intensity from the 5 measurements is added after recording.
To be able to evaluate the data measurements of the scattering from the empty capillary, the capillary containing pure water and the capillary containing buffer must also be recorded. To investigate whether or not the sample will have a large tendency to aggregate at high concentrations the measurements is also executed on samples which are diluted 2 and 3 times respectively. The data that is recorded is the intensity of the scattering on a plate shaped detector. To obtain the intensity as function of scattering vector q = 4πsin(θ)/λ, we integrate the data. Following this step we can subtract the background to obtain the scattering from our particles. To obtain the size distribution function the scattering data is transformed using the indirect Fourier transform
In vitro Dicer assay
5 μL RNA in sulution
2 μL 5x Dicer reaction buffer
3 μL Recombinant Dicer enzyme
Reaction is kept at 37°C over night. NOTE: RNA scaffold is radioactively labeled for analysis.
Dual Luciferase Assay
Preperation of structure
To avoid activation of the interferon immune response when our structure is transfected into cells, we dephosphorylated all 5’-ends on the RNA strands before self-assembly. For the self-assemply we used a molar relation 1:10 between scaffold and staple strands. The dephosphorylation was executed with Antarctic Phosphatase from New England Biolabs, from the manufacture protocol ( http://www.neb.com/nebecomm/products/protocol76.asp). 5 µl scaffold (1 µM), 5 µl of each staple strand (10µM), 1 µl Antarctic Phosphatase, 6 µl Antarctic Phosphatase buffer, incubation for 20 min at 37⁰C, followed by heating to 65⁰C for inactivation of the phosphatase. The self-assembly was done as previously described in total volume of 80 µl containing 1x TAE and 12.5 mM MgAc.
Cell transfection and assay
To the dual luciferase assay we used a H1299 lung cancer cell line with staple integrated reporter gene. The cells were transfected with Lipofectamine2000 in 96 well format with 4 replications of each sample. We made three positive controls, two using siEGFP in concentrations 2nM and 40nM and one with siEGFP-cy3 (40nM). Both have complimentary sequence with the EGFP site in the cell genome. Besides this we made three 3 negative controls, one with siEGFPmismatch (40nM), one with siRNA with non-complementary sequence to EGFP (40nM), with cells only treated with transfection reagent and finally one with not treated cells. The transfection was executed in serum free RPMI (Roswell Park Memorial Institute) media, as we do not know the stability of our structure in serum. We used a cell concentration on 8000 cells/well and we transfected in a total volume on 120 µl, with 100 µl RPMI and 20 µl liposome2000 with siRNA in wanted concentration. We were not able to determine the concentration of our purified structure, but we estimate that the transfection concentration was around 2nM. The non-purified structure was transfected to the cells in concentration around 16nM. 6hours after transfection the cells was washed in PBS (Phosphate-buffered-saline) and 100 µl RPMI with 10% bovine foetal serum was added. The cells incubated at 37⁰C for 2 days before the dual-luciferase assay was executed. The media was removed, cells washed in PBS and 50µl 1x Passive Lysis Buffer was added to each well followed by incubation for 10min. After lysis of cells, 10µl sample from each well was taken to the dual-luciferase assay. Thee assay was executed with “Dual-luciferase reporter assay system” (Promega) using the manufacture protocol. The measured luciferase activities were normalized to the firefly luciferase signal.