iGEM Week 10: Progress Report Triggered Sequestration with DNA Nanostructures: A New Drug Delivery Method August 14, 2006 iGEM Week 10: Progress Report Tiffany Chan, Katherine Fifer, Valerie Lau, Matthew Meisel

Overview Project goal Last week’s progress This week’s goals Test AscI digest Microcon filtration [Mg2+] and [oligo] titration This week’s goals

Project goal To create a scaffolded DNA nanostructure that protects a ligand from degradation When the ligand is presented on the outside of the nanostructure, we expect there to be no protection

AscI digest The “attachment oligo” and the “oligo ligand,” with 15 complimentary bps, were briefly incubated at room temperature The double-stranded construct was then treated with AscI under four different digestion conditions Results: Digestion for 24 min with 500mU of AscI works well. 25 bp+ 10 bp+ 10 bp+ 25 bp+ milliunits AscI: 0 0 0 50 500 0 50 500 digestion time: 0 0 12 12 12 24 24 24 (minutes) 18% PA, 1.5hr @ 120V, SYBR gold staining

Image courtesy Millipore Microcon filtration Samples are pipetted into the Microcon filter and are successively diluted and centrifuged Results: relatively good oligo removal, but poor nanostructure yields Image courtesy Millipore 1 kb+ unpurified successive flow-throughs flowthrough scaffold retentate retentate 2% agarose, 10mM MgCl2, 1hr @ 60V, EtBr

[Mg], [oligos]: PEG experiments 2% agarose, 10mM MgCl2, 1hr @ 60V, EtBr pellet pellet etc… supern. supern. PEG titration performed for each set of folding conditions Results: Oligos were not cleared in any of the 0-8%-final PEG precipitations [MgCl2] - 10mM (final) 20mM (final) 30mM (final) [oligos] 100nm (total) X 600nm (total)

[Mg], [oligos]: Microcon and PEG Lane Trial Purification 1 30 nM oligos, 10 mM MgCl2 unpurified 2 PEG pellet 3 Microcon retentate 4 30 nM oligos, 20 mM MgCl2 Unpurified 5 6 Retentate 7 30 nM oligos, 30 mM MgCl2 8 9 10 1 Kb+ ladder n/a 11 p7308 (44 nM) 12 600 nM oligos, 10 mM MgCl2 13 14 15 600 nM oligos, 20 mM MgCl2 16 17 18 600 nM oligos, 30 mM MgCl2 19 20 1 3 5 7 9 11 13 15 17 19 2 4 6 8 10 12 14 16 18 20 Results: PEG seems to be more efficient at removing oligos Nanostructures in PEG seem to be damaged, gel shifting

Other filtration options Prespin Load sample Centrifuge (large molecules elute first) Microcon use more material—is yield loss constant? use a detergent to loosen nanostructures from membrane (Triton X-100?) Sepharose spin columns large molecules elute first homemade: Datta et al. (2003), Anal. Biochem., 317, p. 284. Clonetech Image courtesy Clonetech

This week’s goals Continue filtration/purification trials AscI digest of folded nanostructure