Triggered Sequestration with DNA Nanostructures

August 21, 2006 iGEM Week 11: Progress Report

Tiffany Chan, Katherine Fifer, Valerie Lau, Matthew Meisel

Overview EM Images Nanostructure Purification (PEG) Enzyme-based protection assay Streptavidin Beads and

Biotinylated Boxes

EM Images (snakes on a grid) c5.0 barrel

(10 nM), 0.7% uranyl formate

Appear to be lining up end to end, probably because of the stain

EM Images

EM Images: the power of DNA

Highly Extensible structures Multimerization - could create dimers,

etc by pairing DNA at the ends of two different barrels

PEG precipitation progress

P S P S U P S P S P S U P S P S P S

P - pelletS - supernatantU - untreated

PEG precipitation progress

Results

30mM MgCl2 w/ 1x oligos seems best

PEG purification seems to be working much better

To be continued

double PEG precipitation to purify away more oligos (first trial unsuccessful)

repetition of precipitations to acquire more purified nanostructures and better images

Microcon trials w/ SDS SDS was added to

the buffer used to rinse Micron tubes

1: 1 kb+ ladder 2: p7308 3: unpurified nanostructures 4: 6hb retentate (did not filter oligos) 5: 6hb retentate, washed w/ 0.1% SDS 6: nanostructure retentate 7: nanostructure retentate, washed w/

0.01% SDS 8: nanostructure retentate, washed w/

0.1% SDS

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Enzyme-based protection assay

Goal: digest away oligo-ligand with AscI

1: 10 bp+ ladder 2: attachment oligo 3: oligo-ligand 4: attachment oligo and oligo-ligand 5: attachment oligo, oligo-ligand, AscI 6: nanostructure w/ inward-facing ligand, AscI 7: nanostructure w/ outward-facing ligand, AscI 8: p7308, AscI 9: nanostructure w/ inward-facing ligand (-AscI) 10: nanostructure w/ no ligand, AscI

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Enzyme-based protection assay (TBE gradient PAGE)

Goal: digest away oligo-ligand with AscI

1: 10 bp+ ladder 2: attachment oligo 3: oligo-ligand 4: attachment oligo and oligo-ligand 5: attachment oligo, oligo-ligand, AscI 6: nanostructure w/ inward-facing ligand, AscI 7: nanostructure w/ outward-facing ligand, AscI 8: p7308, AscI 9: nanostructure w/ inward-facing ligand (-AscI) 10: nanostructure w/ no ligand, AscI

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Streptavidin Beads and Biotinylated Boxes 1) Incubate biotinylated boxes with

streptavidin beads (magnetic or agarose) 2) Wash away unbound material 3) Elute the bound material by digesting

streptavidin with trypsin, proteinase K, or by some other elution measures

Streptavidin Beads and Biotinylated Boxes

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Lane Component1 1kb ladder2 p73083 6hb wash4 biotin-oligos wash5 inside-biotin box wash

6 outside-biotin box wash

7 6hb elute8 biotin-oligos elute 9 inside-biotin box elute10 outside-biotin box elute

Streptavidin Beads and Biotinylated Boxes

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trypsin elution - NO ELUTE QuickTime™ and aTIFF (Uncompressed) decompressor

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streptavidin elution - NO ELUTE

Streptavidin Beads and Biotinylated Boxes Plans:

1) Elute using enzyme-specific buffers 2) Try streptavidin elution using heat or

nanobox-denaturing methods, relying on being able to visualize the ssDNA and oligos

Future

Image folded design 6 Perfect PEG purification Improve protection assays