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Introduction

Paul Rothemund has discovered that it is possible to shape M13-Phage single-strand-DNA simply adding oligonucleotides that will work as „brackets“ when complementing the long single-strand.
In this way, one can generate for example DNA-squares of a certain size with „nods“ at certain distances. One member of our team, Daniel Hautzinger, has recently finished his diploma-thesis on Origami-DNA and the possibilities of generating patterns on these square surfaces by modifying the oligo-nucleotides that build up the nod-points.
As the antigens NIP and fluoresceine can as well be fused to these oligos, we had found the seemingly perfect tool to present strictly defined two-dimensional antigen-patterns to cells carrying our synthetic receptor system. File:Freiburg2008 NIP arrangement schematical.jpg

Literature

• Paul W. K. Rothemund: Nature 440, 297-302 (16 March 2006)

Methods

Phage DNA

Cell culture

50 ml DYT-Medium, 50µl tetracycline (TET; 25 mg/ ml) and ER2738-cells were shaken over night at 37°C. The overnight culture was diluted with DYT to OD600=0.1 and shaken at 37°C until the culture had an OD600 around 0.4. Each 50 ml of cell culture were inoculated with 5 µl M13mp18 phage and shaken for 4 h at 37°C.

Isolation of M13mp18 phage from cell culture

PEG/NaCl was used to precipitate the phages.
First precipitation
Each 50 ml of cell culture were centrifuged at 5000 g for 20 min. While the cell were centrifuged 1/7 volume of the supernatant PEG/NaCl (about 7ml/50ml) was put in a new falcon tube. The phages stay in the supernatant therefore the supernatant was carefully decanted to the PEG/NaCl and mixed gently by inverting the tube. The mixture (Solution 1) was left overnight at 4°C.
Solution 1 was centrifuged at 5000 g for 20 min. Because the phage stay in the pellet, the supernatant was removed and the pellet was resuspended in 2 ml TBS-Buffer (Solution 2). Solution 2 was put in a 1,5 ml Eppendorf tube and centrifuged (13200 rpm, 10 min). After the centrifugation the phages stay in the supernatant.
Second precipitation
170 µl PEG/NaCl(~ 1/6 volume of supernatant) were put in a Eppendorf tube. Supernatant was carefully decanted to the PEG/NaCl and mixed gently by inverting the tube. The mixture (Solution 3) was left for 1 h on ice. Solution 3 was centrifuged at 13200 rpm for 10 min.

Define phage titers

The absorption of Solution 3 was measured on a Jasco V-550 UV/VIS spectrometer at 269 nm.
Phage titer was calculated as follows:
Phage DNA = ((A269-A360) * 6 * 10^16 * dilution factor) / (number of bases in the phage genom = 7249 bp)

Isolation of the phage DNA

The phage DNA was isolated with QIAprep Spin M13-Kit (50) from QIAGEN (Cat.No: 27704).
DNA-concentration was quantified by Nano-drop photometer.

Origami

Produce the Origami

To produce the Origami we mixed each the M13mp18 DNA with the oligos, water and TEA/MgAcetat (end concentration =12.5mM).

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<o:p> TABELLE 1</o:p>

<o:p>TABELLE 2</o:p>

Various samples were produced. For the sample with a ratio of 1:20 (DNA:Oligo) we used 4 nM DNA and 80 nM of oligos. The Origami were produced in a eppendorf Mastercycler personal. Therefore they were heated up to 95°C for 7 min and slowly cooled down (0.3°C/s) to 20°C.
Different sample were made:
Sample 1:20, 1:10 and 1:5 without NIP, oligos without NIP were used
Sample 1:20, 1:10 and 1:5 with NIP, all of the 7 oligos with NIP were used -> origami with 7NIP
Sample 1:5 with NIP and fluorophor, all of the 7 oligos with NIP and the 2 oligos with the Alexa 488 were used.