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To check the yield of the annealing reaction, 2.5 pmol of the annealed duplex were run on a 4 % aggarose gel.
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===
====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) [insert link to purification].   
A plate was folded using the standard protocol (insert link to plate folding) and purified on an Amicon 100K spin filter (Millipore) [insert link to purification].   
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 M13 DNA.
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 M13 DNA.

Revision as of 11:32, 19 October 2013

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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), 1 µL of PepLock staples (5 µM), 1 µL ofcholesterol-ModRight1 staples (5 µM), 1 µL of cholesterol-ModRight2 staples (5 µM), 1 µL of cholesterol-ModRight3 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 37oC for 30 minutes, and subsequently stopped by adding 1 µL 0.5 M EDTA and incubating the reaction at 75oC 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 95oC to 4oC 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) [insert link to purification]. 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 M13 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 4oC, 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 37oC to allow the overhang of the sisiRNA to anneal to the staple strands of the plate.

Luciferase assay

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.

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</style> </head> <body> <div id="indexing"> <div id="sitemap"> <p id="sitemapTitle">SITEMAP | BIOMOD 2013 NANO CREATORS | Aarhus University</p> <div id="footer-contents"> <div class="footer-section"> <p class="footer-section-title">INTRODUCTION</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus">Home, abstract, animation and video</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Introduction">Introduction</a></li </ul> </div> <div class="footer-section"> <p class="footer-section-title">RESULTS AND DISCUSSION</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/Origami">Origami</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/Peptide_lock">Peptide lock</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/Chemical_Modification">Chemical modification</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/sisiRNA">sisiRNA</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/System_In_Action">System in action</a></li> </ul> </div> <div class="footer-section"> <p class="footer-section-title">MATERIALS AND METHODS</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Origami">Origami</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Peptide_lock">Peptide lock</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Chemical_Modification">Chemical modification</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/sisiRNA">sisiRNA</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/System_In_Action">System in action</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Methods">Methods</a></li> </ul> </div> <div class="footer-section"> <p class="footer-section-title">SUPPLEMENTARY</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/Team_And_Acknowledgments">Team and acknowledgments</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/Optimizations">Optimizations</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/Supplementary_Data">Supplementary data</a></li> 

                                               <li><a

href="/wiki/Biomod/2013/Aarhus/Supplementary/Supplementary_Informations">Supplementary informations</a> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/References">References</a></li> </ul> </div> </div> <div> <p id="copyright">Copyright (C) 2013 | BIOMOD Team Nano Creators @ Aarhus University | Programming by: <a href="mailto:pvskaarup@gmail.com?Subject=BIOMOD 2013:">Peter Vium Skaarup</a>.</p> </div> </div>

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