IGEM:Harvard/2006/DNA nanostructures

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Contents

Project Overview

  • Our goal is to to design and implement molecular containers, which can be dynamically opened and closed by an external stimulus.
  • The containers will be implemented as DNA nanostructures, which afford a significant degree of positional control and chemical versatility.
  • As an initial proof-of-concept, we plan to use our DNA containers to demonstrate controllable activation ("delivery") of anti-thrombin aptamers.
  • We expect that molecular containers could have several interesting scientific and clinical applications, such as
    • Drug and gene delivery
    • Bio-marker scavenging (early detection of biomarkers)
    • Directed evolution (compartmentalized selections)
    • Using multiplexing for combinatorial chemical synthesis
    • Capture and stabilization of multiprotein complexes
    • Protein folding (chaperones)
    • Cell sorting

Container Specs

Image:iGEM_harv06_mattspecs.gif

Container Designs

Latch Designs

Coding

Existing code

Thrombin-aptamer experiments

Notes

Questions / procedures

  • what percent gel? 10% to 20% polyacrylamide gels, no SDS (but would make for a good control)
  • what incubation conditions?
  • how much protein and DNA? protein at 1 μM, DNA at 2 μM
  • Coomassie stain

Experiments

number thrombin aptamer nanotube DNA-stained prediction protein-stained prediction
0---no bandsno bands
1--+slow band (nanotube)no bands
2-+-fast band (aptamer)no bands
3-++slow band (aptamer-nanotube), traces of fast band (aptamer)no bands
4+--no bandsfast band (thrombin)
5+-+slow band (nanotube)fast band (thrombin)
6++-medium band (aptamer-thrombin), fast band (aptamer)medium band (aptamer-thrombin), traces of fast band (thrombin)
7+++very slow band (thrombin-aptamer-nanotube), slow band (aptamer-nantotube), traces of fast band (aptamer)very slow band (thrombin-aptamer-nanotube), medium band (aptamer-thrombin), traces of fast band (thrombin)

Buffers

  • Macaya's and Bock's selection buffer: 20 mM Tris-acetate, pH 7.4, 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2
  • Liu's incubation buffer: 40 mM Tris, 20 mM CH3COOH, 2mM EDTA, 12.5 mM (CH3COO)2Mg, pH 8.0
  • Liu's PAGE buffer: 1x TAE/Mg2+

Potential procedure for 10 μL incubation reaction

  • In 1 0.2 mL PCR tube, mix:
    • 2 μL of 5x Bock's selection buffer
    • 2 μL of 10 μM scaffold DNA (final concentration: 2 μM = 20 pmol)
    • 2 μL of 10 μM oligos (final concentration: 2 μM = 20 pmol)
    • 2 μL of 10 μM aptamers (final concentration: 2 μM = 20 pmol)
    • 2 μL of 5 μM thrombin (final concentration: 1 μM = 10 pmol)
  • Alternative mix: Liu uses 10 pmol (1 μL of 10 μM) and varies thrombin from 2 pmol (1 μL of
  • Incubate at room temperature for 30 min.
  • Load onto a non-denaturing polyacrylamide gel (10% to 20% gradient)
    • Liu runs at 25 mA for 48 h.

Bibliography

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  2. Error fetching PMID 15945116: [tha2]
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  5. Error fetching PMID 8475124: [tha5]
All Medline abstracts: PubMed HubMed

Presentations

Most recent (Week 3)

Week 2: Original proposal

Working Team Members

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