Team:TU Eindhoven/Mechanism

Step 1

For the Rolling Circle Amplification to be used on the surface of cells a DNA-primer has to be covalently linked to the cells. This primer can then be used to start the amplification of the DNA-strand to form the long strands needed for Multiple Chain Amplification. In order to attach DNA to the cell surface, the DBCO-Azido click system developed was used. The step described focusses on attaching a short DNA oligo to a DBCO-PEG4 linker molecule. This is the building block that is used to click DNA to the cells and start a Rolling Circle Amplification reaction.

In order to attach DNA-primers to a cell the click-system developed by Team Eindhoven 2014 can be used. For this to work the primer needs to be functionalized with a DBCO-group. This was done by attaching a DBCO-PEG4 molecule to the oligo required for Rolling Circle Amplification. To attach the molecules which normally don’t react together, DBCO-PEG4-NHS ester was ordered as well as a 5’-amine with a twelve carbon spacer functionalized primer. These two molecules where reacted for two hours at room temperature to form the product.

This reaction was carried out at room temperature for two hours after which it was analyzed on a 15% PAGE gel. On the gel it clearly showed that the product after two hours was heavier, thus indicating that DNA had reacted with DBCO-PEG4-NHS ester.

The final step was to purify the product and remove any unreacted DBCO-PEG4-NHS ester. This was done through ethanol precipitation of DNA. This was done once after which concentration in 100 µL Mili Q water was determined. The final concentration was used to determine the yield of this reaction step and was around 65%, including washing. The resulting molecule can be used to click DNA to the cell surface which was used to initiate a rolling circle amplification reaction used to create a DNA-based hydrogel around the cell.

Step 2

Once the DBCO-primer product is constructed, there is a need to validate that it is functional. To do so, two functions of the DBCO-primer have to both be tested and shown to function, the ability of DBCO to click to azide-groups in COMPx and the DNA to form hydrogen bonds with complementary DNA. Both of these were evaluated in a single step in which the product was reacted with cells expressing COMPx and later labelled with a fluorescent DNA-probe. This was then put in the FACS machine to determine the difference between unlabeled cells, cells with the primer clicked to the surface and cells labeled with DBCO-PEG4¬-5/6TAMRA. From these results it was concluded that the product still functioned as expected.

The previously created DBCO-PEG4-primer has to be tested in order to determine if it’s still functioning, thus making sure it can be used as a starting point for the Rolling Circle Amplification. To test this, cells expressing the COMPx protein where cultured and filtered from 2YT medium by down spinning. The cells were then resuspended in cold PBS so to stop cell division. All samples were then diluted to a concentration of 109 cells/mL. One sample 200 µL was then kept on ice and not reacted any further, to serve as a negative control. One 200 µL sample was reacted with DBCO-PEG4-5/6TAMRA for 1 hour at 4°C and 500 rpm to check the effectiveness of the primer compared to a positive control. The last 200 µL sample was reacted with the DBCO-PEG4-primer for 1 hour at 4°C and 500 rpm. The last two samples were then isolated from PBS by down spinning once again. The sample labelled with primers was then incubated with a complementary fluorescent primer for 1 hour at 4°C and afterwards the fluorescent probe was removed by spinning the cells down. All three samples were then run through the FACS machine to test for fluorescence. The graph below indicates that while the fluorescent primer didn’t give the same shift in measured fluorescence as the positive control it did clearly bind to the complementary probe which was clicked to cells thus showing that the product formed was still fully functional.