03.09.2012

Main topic: Task distribution for the week. Upon discussion with the Aptamer Team (Ali and Maryam) and Praveen, the following tasks were scheduled to be done during this week:

• Do a chart comparing the prices, delivering times, characteristics for the PDGF that the different companies offer. (Aptamer Group)
• Contact Ralf Seidel (Seidel’s group has stuff from Sigma, so they might have some discount or partnership) or Sigma-Aldridge directly, as they have Human PDGF BB in their stock, and ask for a sponsorship or a discount for the team. (Ali is working on it). (Aptamer Group)
• Order the optimal PDGF (AS SOON AS POSSIBLE!!-> The delivery times are around 2 weeks!!!). (Aptamer Group)
• Ask Michael Schlierf if an spectrophotometric assay might be the best technique for determining the reaction kinetics of the aptamer in labeled and unlabeled conditions. If it is; start right away as soon as the PDGF is here!!! If not, then ask him what he would suggest (FRET?)(Aptamer Group)
• Contact Dominik to ask about the new chol modified sequences that Yixin sent and confirm with Alex and Santiago if they do not overlap with the strands in the origami structure. (GUVs and Origami Group)
• In case they overlap, they have to be immediately ordered from a company (no time for synthetizing them) using sequences that do not overlap for sure. (GUVs Group)
• Ask Dominik and Ralf about the Quantum Dots usage, assemble new structures with QDs and start running the respective experiments; LSM for the GUVs and Gel Shift for the Origami. (GUVs and Origami Group)
• TEM image all the different DNA Origami constructs that were assembled to determine the best conditions for the origami and for negative staining. (Origami Group)
• Determine the Layout and colors of the wiki webpage. Discuss the logo. (Santiago)
• Meet Marino Zerial at 3:00pm tomorrow (05.09.2012) to discuss about possible applications for the project. IMPORTANT: The feedback we get from Marino should be used to write the ABSTRACT of the project!!!!!! The more or less final draft has to be done the latest on Friday, so that next week on Monday we can meet with our supervisors for final corrections!!!!!! REMEMBER!!! ---> The abstract has to be send before Friday 14.09.2012 to shawn.douglas@wyss.harvard.edu. (Everyone).
• Next Meeting on Friday 07.09.2012 before Beer Hour in Biotec (16:00). Results/progress evaluation!!

31.08.2012

Summary

• The biotin labelled oligos arrived.
• The aptamers and complements arrived.
• The labelling was done till today as the biotin labelled oligos arrived till wednesday
• Ask Yixin Zhang about the cholesterol labelled oligos->How does it work? could we do it? also discuss TEG spacer with him.

REMARKS/COMMENTS:

• Option given by svea->Polystyrene beads (1um) if QDs don't work.
• Keep in mid Articles in German for enterprises.

IMPORTANT TASKS: To order (Everybody should be aware):

• Catcher complementary + chol @ 3'->To connect LUVs
• Anchor complementary + chol @ 3'->To connect the DNA origami with the GUVs IMPORTANT!!!!!

To do:

• Clarify the procedure for chol labelled oligos and the TEG spacer -> Yixin Zhang Cost and time are key!!!! (Praveen)
• Talk with the technician (or someone) that know about the process. To be done on monday. (Praveen)
• Get PDGF. Santiago (me) will insit with MP Biomedicals. But please-> Look for other companies and harrass them if it's necessary!!(Ali and Maryam)
• Start aptamer experiments right away. Characterize aptamer and test the locks->Does labelling affect/change function? (Ali and Maryam)
• Do GUV-QD exps. during this week-> Attach complementary stands to GUVs and QDs and do microscopy (Varsha, Praveen)
• Assemble new constructs and do Origami-QD exps-> Gel shift and TEM (Alex, Santiago)
• Advance with the wiki!! -> Define a solid layout (Santiago)

20.08.2012

Fahrgarten Review and remarks from 17.08.2012

• Check the size of the construct vs. the GUV’s size. Do they fit?, What about QDs
• Where aptamers ordered with BHQs?
• General stuff about Visa procedures->Transport, bus/No telephones or handpack
• T-shirts and logo should be designed probably using Dresden skyline in a comic version (siluette)

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.