Biomod/2013/Aarhus/Materials And Methods/System In Action

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System in action

Assembly of origami baseplate with cholesterol

2 µL of MgCl2 (125 mM) reaction buffer was mixed with 1 µL of M13mp18 (418 nM). 1.5 µL of BG1 staples (3.55 µM), 1.5 µL of BG2 staples (3.55 µM) and 1.5 µL of BG3 staples (3.55 µM) were added, followed by 1 µL of Dome staples (5 µM) and 1 µL of photosensitizer-ModLeft staples (5 µM). 6.5 µL of 0.5× TAE was added to a total volume of 20 µL. The reaction was subsequently incubated for 17 hours in a PCR machine with a non-linear time ramp and purified with 100 kDA Amicon filters.

Assembly of origami baseplate with photosensitizer

In order to assemble our origami baseplate with the photosensitizer-modified staple strands, 2 µL of MgCl2 (125 mM) reaction buffer were mixed together with 1 µL of M13mp18 (418 nM) (scaffold strand). 1.5 µL of BG1 staples (3.55 µM), 1.5 µL of BG2 staples (3.55 µM) and 1.5 µL of BG3 staples (3.55 µM) were added, followed by 1 µL of Dome staples (5 µM), 1 µL of PepLock staples (5 µM), 1 µL of 5 µM ModRight staples and 1 µL of photosensitizer-ModLeft staples (kan henvises til Mors påfund) (5 µM). 8.5 µL of 0.5× TAE were added to a total volume of 20 µL. The reaction was subsequently incubated for 17 hours in a PCR machine with a non-linear time ramp and purified with 100 kDA Amicon filters

Labeling of staple strands with terminal transferase

A master mix consisting of 26 µL 5× TdT reaction buffer (Roche), 26 µL CoCl2 (25 mM), 6.5 µL TdT Stock and milliQ H2O to a total volume of 130 µL was prepared. 1 mM Cy3-ddUTP or 1 mM Cy-5-ddUTP added to the mix. For the TdT-reactions with Cy-3-ddUTP, 13 µL staples of Modleft (10 µM) was added and in the TdT-reaction with Cy-5-ddUTP, 13 µL staples of ModRight/Purple (10 µM) were added. The reactions were incubated at 37˚C for 40 minutes and subsequently stopped by adding 13 µL EDTA (0.2 M).

Staining the samples for confocal microscopy

KB-EGFPluc-Wagner were seeded out as 8x104 cells/well in an eight well chamber slide. 24 hours after seeding out, the cells were transfected with the origami plate with attached cholesterols to a final concentration of 0.5 nM origamis baseplate for 2 hours. The medium was removed and the cells were washed twice with PBS buffer with 1 % sodium azide. The cells were then stained with 5 µg/mL Wheat Germ Agglutinin, Alexa Fluor 488 Conjugate (WGA-A488) (Life Technologies) for 10 minutes at 37˚C. The cells were washed twice in PBS buffer with 1% sodium azide. Fixation was done by adding 4% PFM and left for 15 minutes at room temperature to fixate. PBS buffer was used to wash the cellw once again. The well cage and gasket from the chamber slide were removed and dipped in a tube of ddH2O to remove salt. The slide was left to air dry for 20 minutes until the sample was approximately 90 % dry. 5µl/cm2 of Prolong Gold with DAPI (Life Technologies) were added on the slide. A cover glass was placed on the edge of one side of the slide. The slide were incubated until the next day in the dark room at room temperature. As the cells could not be visualized until some time later, the slide was treated with nail polish and stored at 4˚C.

Melittin modified sisiRNA attached to the origami plate

20 pmol of W376 was labeled using a T4 polynucleotide kinase kit (Thermo Scientific) following the pipetting scheme below.

Radioactive labeling of W376 2 µL 10 x reaction buffer A 20 pmol γ-32P-ATP 1 µL T4 polynucleotide kinase (10 U) 20 pmol W376 RNase free water to a total reaction volume of 20 µL.

The reaction was incubated in the PCR machine at 37°C for 30 minutes, and subsequently stopped by adding 1 µL 0.5 M EDTA and incubating the reaction at 75°C for 10 minutes. The reaction was then purified on a Sephadex G-25 spin column (GE Healthcare).

Annealing reaction

The labeled W376 was annealed with a 1.3 times excess of the two segments of the passenger strand, W004 and W179 in annealing buffer containing 200 mM potassium acetate in 20 mM HEPES (pH 7.4) and incubated in the PCR machine with the temperature linearly decreasing from 95°C to 4°C over 1.5 hours. To check the yield of the annealing reaction, 2.5 pmol of the annealed duplex were run on a 4 % aggarose gel.

Attachment of sisiRNA to the origami plate

A plate was folded using the standard protocol (insert link to plate folding) and purified on an Amicon 100K spin filter (Millipore). A sample containing ~ 65 . 10-15 of the purified plate was run on a 1 % aggarose gel containing 7 µL SYBR safe (Invitrogen) per 100 ml gel and 5 mM MgCl2 along with a sample of the non-purified plate and a control of M13mp18 DNA. The labeled sisiRNA duplex was annealed to the plate by mixing the plate with a three times excess of labeled sisiRNA per binding site, i.e. a 30 times excess per plate, as each plate contains 10 binding sites, followed by 20 minutes of incubation at 37oC. The sample was the run on a 1 % aggarose gel for 3 hours at 110 V , 4 W at 4°C, and the placed in a storage phosphor screen (GE Healthcare) for development over night. Approximately 10 hours later the phosphor storage screen was scanned on the Typhoon scanner using phosphor storage mode. To verify that the uppermost bond did correspond to a folded plate, the gel was stained with SYBR Gold and scanned.

Transfection with melittin conjugated sisiRNA attached to the origami plate

A doubly modified duplex with W004-melittin and W179-melittin were annealed to W376 and run on a 4 % aggarose gel to estimate the yield of the annealing. A batch containing 15 pmol of plate was prepared and purified on a spin filter and checked on a 1 % aggarose gel. The purified plate was mixed with a 30 times excess of the sisiRNA duplex and incubated for 20 minutes at 37°C to allow the overhang of the sisiRNA to anneal to the staple strands of the plate.

Luciferase assay

For the luciferase and MTT assays, the cells were transfected with the doubly melittin-modified sisiRNA attached to the origami plate in eith two different concentrations of sisiRNA, 50 nM and 10 nM. As each plate contains 10 binding sites for the sisiRNA, the concentration of the origami plate was 5 nM and 1 nM in these samples. For each concentration two transfections were performed, one with Lipofectamine and one without it.


Test system in action

Figure 69: Schematical overview of test system DII
Figure 69: Schematical overview of test system DII

Automated oligonucleotide synthesis was employed in the synthesis of 3'- and 5' amino modified DNA strands. These strands was used in the setup shown on Figure Test. The 3’amine was set to use universel support with DMT on and a 3’amine Mod C7 CPG 1000 Mermade column from link technologies. The 5’amine was set to use standard support with DMT off and a 5-MMT- AminoMod C6-CE Phosphoramidite. After completion of the synthesis, DNA was cleaved from the solid support through incubation with AMA (0.5 mL) at 65°C for 30 mins.

The synthesized 5’amine DNA strand was evaporated and redissolved in MQ water (200 μL). The liquid was transferred to an Eppendorf tube (thus removed from the column material). The DNA was precipitated in ethanol and lyophillized. The DNA pellet was redissolved in MQ (101 μL)

The synthesized 3’amine modified DNA strand was purified by TOP column chromatography: NaCl (0.5 mL 100mg/mL) was added to the sample . The column was prepared for chromatography by adding MeCN (0.5 mL) and TEAA (1 mL, 2M) and running the liquid through. The sample was transferred to the column and washed with a NaCl (2mL, 100 mg/mL) solution. The DNA was detritylated with TFA (21 mL, 5 % aq). It was observed that the column turned red. The DNA was rinsed with water and the column became colourless. The sample was collected from the column with MeCN/MQ 1:1 (1mL and then 0.5 mL). The product was evaporated and redissolved in MQ (101 μL).

Conjugation of the synthesized 3’amine modified DNA strand with In(PPa-NHS ester)Cl

A general procedure for conjugating an NHS ester with an amine modified DNA strand was followed. [1] In(PPa -NHS ester)Cl (5 mg) was dissolved in dry DMSO (100μL) and added to a tube with DNA (142 nmol, 100 μL). Precipitate was observed and MeCN (3x100 μL) was added. TEA (5 μL) was added and the reaction incubated at 23 ⁰C o.n. The DNA was precipitated with NaOAc (43 μL), glycogen (1 μL) and cold ethanol (750 μL). The mixture was incubated on dry ice for 15 min and centrifuged (4⁰C, 60 minutes, 20817 rcf). The supernatant was decanted off and the DNA pellet redissolved in TEAA buffer (300 μL). The crude product was purified by RP-HPLC (10% to 70% MeOH in TEAA buffer over 30 min). The product was lyophilized o.n.

Conjugation of the synthesized 5’amine with the NHS ester of cholesterol

A general procedure for conjugating a NHS ester with an amine modified DNA strand was followed. [1] The NHS ester of cholesterol (3mg) was dissolved in dry DMF (75 µL) and the 5’ amine modified DII DNA (72 nmol, 75 µL) was added. Precipitation was observed and MeCN (3x75 μL) was added. The reaction mixture was incubated at rt over 3 days. The mixture was shortly centrifuged, the supernatant was removed and the precipitate was discarded. To precipitate the DNA NaOAc (54 µL), glycogen (1 µL) and cold ethanol (750 µL) were added. The mixture was incubated on dry ice for 15 min, centrifuged (4 ⁰C, 45 minutes, 20817 rcf), and the DNA pellet was redissolved in TEAA buffer (0.1 M, 200 myL) and purified by RP-HPLC (10% to 70% MeOH in TEAA buffer over 30 min). The product was lyophilised o.n. and redissolved in MQ (100 µL).

PAGE analysis; annealing of test system

Prior to the gel the samples were annealed by heating to 75°C degrees, holding this temperature for 5 minutes and then slowly cooling to rt.

In all wells were added: Buffer (2 μL, NaCl (2M)/ HEBES (0.5 M, pH=7.5) 1:1), loading buffer (2 μL, 6x sucrose orange g) and MQ to give a total volume of 10 μL. The DNA samples added contained 5 pmol each. The ladder used was O’gene Ruler Ultra Low Range DNA ladder (2µL) (25 bp ladder) The gel was a native gel consisting of 4mL tricine, 6 mL acrylamide (40%), 30 mL MQ water 30 mL, 40 µL TEMED and 400µL 10% APS. Three different designs had been tried in order to get a good test system where the modified DNA strands annealed properly. The chosen design was design DII. The gel was forerun for 30 minutes, loaded and run for 2½h. The gel was scanned on the typhoon scanner to see the In(PPa-Cl) prior to staining. The gel was stained with ethidium bromide for 10 minutes and scanned for the DNA strands

Cell experiments

The cell experiments was carried out in collaboration with Thomas Breitenbach (Center for Oxygen Microscopy and Imaging, Department of Chemistry and iNANO, Aarhus University), who also provided the HELA cells.

The ability of DII to kill cells was tested on HELA cells (seeded out one day prior to the experiments, density ~ 75,000 cells/ml). The cells were incubated in a HEBES-buffered media AMB (artificial bath medium, pH=7.4.) All samples for the cell experiments was prepared in low-bind Eppendorf-tubes in ABM buffer to a total volume of 500 µL All samples were incubated at 65ºC for 10 min before incubation of the cells.

Incubation procedure: The coverslip was removed from the growth medium, placed in a clean well and rinsed with ABM buffer (2*500µL). The sample was added and the cells left for a certain amount of time. The coverslip was removed to a cuvette and rinsed with ABM buffer (2·500µL). AMB buffer (500µL) was added.

Since DII is very weak fluorescent a new design DII* was made to see how the test system interacted with the cells.

Conjugation of 3’amine modified DNA strand with PPa-NHS ester (design DII*)

PPa -NHS ester (2 mg) (provided by Thomas Tørring) was dissolved in dry DMSO (100 μL) and added to an Eppendorf tube with 3’amine modified DNA strand (23 nmol, 100 μL). Precipitate was observed and MeCN (3*100 μL) was added. Triethylamine (5 μL) was added and the reaction incubated at 23 ⁰C for 3h. The DNA was precipitated with NaOAc (71 μL), glycogen (1 μL) and cold ethanol (1250 μL). The mixture was incubated in the freezer o.n. and centrifuged (4 ⁰C, 45 minutes, 20817 rcf). The supernatant was decanted off and the DNA pellet redissolved in TEAA buffer (200 μL, 0.1M). The crude product was purified by RP-HPLC (10% to 70% MeOH in TEAA buffer over 30 min). The product was lyophilized o.n. and redissolved in MQ (50 µL).

References

  1. Jahn, K. et al. Functional patterning of DNA origami by parallel enzymatic modification. Bioconjugate Chem. 22, 819–823 (2011).[1] [Jahn]

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