17.08.2012

General description of the aptamer.

• Send a mail to Shawn Douglas to check the consistency of the ordered aptamers.
• Is it the same to label the 3’ with Cy3 and 5’ with BHQ as doing the other way around? The company sponsoring the aptamers suggests labeling on 3’ with the dye.
• Around the last ~15 bases of the aptamer are the ones that actually react with the protein during the aptamer conformation change.
• Check where we can buy the PDGF. How much does it cost? I checked and it costs 254 euros for 25ug and 7741 for 10mg
• Check how the aptamer labeling works?
• Check the kinetics of the aptamer -> How quick is the aptamer -> Measure speed of aptamer, having quencher and fluorophore.
• Fluorophore and quencher are around 1~1.5 nm.
• Can actually one make the sequence longer without altering the functioning of the aptamer?
• NOTE: Ask if they actually desisted of doing the aptamer labeling because of its difficulty
• Ralph suggest that we test opening and close by binding. Why bothering with the specific lock closing, if the proof of concept is binding?
• QD to prove that 655nm QD->15nm in case that the LUVs doesn’t work QD
• Gel shifting upon the binding of QDs -> Nice way of probably testing the binding of QDs .
• Additionally CCS (Cross Cor. Spec) can be performed also.
• Probably we don’t need both locks to prove the concept.
• Kinetics of the aptamer is ok to show. Prove and recook.
• Risky strategy suggested by Michael . Order the 4 oligos (aptamers and complements) and add to additional aptamers with the 3’
• PAGE gel with the labeled oligo and see difference between hybridized and non hybridized structures.
• Heating up the sample during the assembly process
• Run a simple hybridization process Heat and cool, observe fluorescence changes in time. Leave it in the Fluor. Spectrometer -> We can do it in B CUBE, they

DNA Origami

• Run the gel with QDs for the origami. Biotin attaching to the 5’ ends of the catcher strands and connected to QDs. Begin with a central strand -> check for a shift in a gel. Test hybridization. Order additional linkers through Sygma, the linkers also Biotin labeled to test the hybridization. Basically QD replace the LUV.
• Santiago’ll do negative staining with Susanne Kretschmar (ask for the proper preparation time witout biasing her).

Summary/Discussion with supervisors

• Ask Douglas about the “mysterious” TTs and the Mg++ solution.
• Run a control for GUV-> Have a GUV coated with strands and try to bind the QDs with the complementary bps. If the QDs work we can go for the origami.
• Next step would be to check the specific attachment of the DNA origami with the GUVs. Probably
• The proportion of biotinated oligos should be slightly over 1:1, though 1:1 is ok.
• Purify out (flush out) the linkers so that the single ones do not interfere by binding to the LUVs
• Ask Dominik to order QDs and Biotin.
• Order oligos and Biotins and run experiments both in GUV and DNA origami side!!!
• Contact the following company for asking about sponsorship and to order the PDGF MP Biomedicals GermanyToll Free Tel: 00800 7777 9999 Tel: 0800-426 67 337 Toll Free Fax: 0800-629 67 337 Email: custserv.de@mpbio.com
• Prepare the aptamer sequences and order them:
• Run some kinetics on the aptamers alone-> Nice to show in results.