Triggered Sequestration with DNA Nanostructures

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Triggered Sequestration with DNA Nanostructures

• August 21, 2006
• iGEM Week 11 Progress Report
• Tiffany Chan, Katherine Fifer, Valerie Lau,
  Matthew Meisel

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Overview

• EM Images
• Nanostructure Purification (PEG)
• Enzyme-based protection assay
• Streptavidin Beads and Biotinylated Boxes

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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

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EM Images
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EM Images the power of DNA

• Highly Extensible structures
• Multimerization - could create dimers, etc by
  pairing DNA at the ends of two different barrels

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PEG precipitation progress
P S P S U P S P S P S U
P S P S P S
P - pellet S - supernatant U - untreated
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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

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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

1 2 3 4 5 6 7 8
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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

1 2 3 4 5 6 7 8 9 10
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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

1 2 3 4 5 6 7 8 9 10
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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

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Streptavidin Beads and Biotinylated Boxes
1 2 3 4 5 6
7 8 9 10

• Lane Component
• 1 1kb ladder
• 2 p7308
• 3 6hb wash
• 4 biotin-oligos wash
• 5 inside-biotin box wash
• 6 outside-biotin box wash
• 7 6hb elute
• biotin-oligos elute
• inside-biotin box elute
• 10 outside-biotin box elute

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Streptavidin Beads and Biotinylated Boxes
trypsin elution - NO ELUTE
streptavidin elution - NO ELUTE
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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

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Future

• Image folded design 6
• Perfect PEG purification
• Improve protection assays