1 / 9

Triggered Sequestration with DNA Nanostructures: A New Drug Delivery Method

This project focuses on the creation of a scaffolded DNA nanostructure designed to protect a ligand from degradation during drug delivery. The approach utilizes an "attachment oligo" and "oligo ligand" that form a double-stranded construct, which is tested under various digestion conditions using AscI. Progress includes successful AscI digestion results and preliminary Microcon filtration tests. Though oligo removal was effective, nanostructure yields were suboptimal. This week's goals involve further purification trials and digestion analysis to enhance efficiency in drug delivery.

denis
Télécharger la présentation

Triggered Sequestration with DNA Nanostructures: A New Drug Delivery Method

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 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

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

  3. 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

  4. 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

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

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

  7. [Mg], [oligos]: Microcon and PEG 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

  8. 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

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

More Related