IGEM:Harvard/2006/DNA nanostructures

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

Container Designs

• 

  Design 1
  hexagonal core, separate 1-ply lids

• 

  Design 2
  hexagonal core, separate 2-ply lids

• 

  Design 3
  rectangular core, continuous 1-ply lids

• 

  Design 4
  hexagonal core, separate 1-ply lids

Latch Designs

• 

  ai
  

• 

  ai
  

Coding

Existing code

• William's code (Python)

Thrombin-aptamer experiments

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 bands	no 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 bands	fast 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⁺

Protocols

Potential protocol for a 2 μL incubation reaction (revised with Dr. Shih's suggestions)

• Reconsitute lyophilized bovine thrombin
  • biuret is 745 NIH units = 637 μg (1170 NIH units = 1 mg)
  • "A suggested concentration for preparation of a stock solution is 100 units/ml. The solution should contain approximately 0.1% BSA for stability and is stable for about one week at 0-5 °C. Since thrombin solutions adsorb to glass, it is recommended to aliquot the solution in plastic tubes and store at -20 °C or below." [1]
  • Reconstitute 745 NIH units in 0.49 mL 1% BSA and 4.41 mL water (4.90 mL total) to give a working stock of 152 units / mL = 130 μg / mL = 2 nmol / mL = 2 μM (formula weight is approximately 65 kDa [2])
• In a 0.2 mL PCR tube, mix:
  • 0.5 μL of 4x (not 5x) Bock's selection buffer
  • 1.0 μL of 2 μM aptamers (final concentration: 1.0 μM = 2 pmol)
  • 0.5 μL of 2 μM thrombin (final concentration: 0.5 μM = 1 pmol)
• OR in a 0.2 mL PCR tube, mix:
  • 0.5 μL of 4x (not 5x) Bock's selection buffer
  • 0.5 μL of 2 μM aptamers (final concentration: 1.0 μM = 1 pmol)
  • 1.0 μL of 2 μM thrombin (final concentration: 0.5 μM = 2 pmol)
• Alternative mix: Liu uses 10 pmol of DNA (1 μL of 10 μM) and varies thrombin amount from 2 pmol (1 μL of 0.2x thrombin working stock) to 100 pmol (1 μL of 10x thrombin working stock)
• 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.

Matthewmeisel 11:11, 11 July 2006 (EDT)

Bibliography

1. Schultze P, Macaya RF, and Feigon J. Three-dimensional solution structure of the thrombin-binding DNA aptamer d(GGTTGGTGTGGTTGG). J Mol Biol. 1994 Feb 4;235(5):1532-47. DOI:10.1006/jmbi.1994.1105 |
2. Liu Y, Lin C, Li H, and Yan H. Aptamer-directed self-assembly of protein arrays on a DNA nanostructure. Angew Chem Int Ed Engl. 2005 Jul 11;44(28):4333-8. DOI:10.1002/anie.200501089 |
3. Li WX, Kaplan AV, Grant GW, Toole JJ, and Leung LL. A novel nucleotide-based thrombin inhibitor inhibits clot-bound thrombin and reduces arterial platelet thrombus formation. Blood. 1994 Feb 1;83(3):677-82.
4. Bock LC, Griffin LC, Latham JA, Vermaas EH, and Toole JJ. Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature. 1992 Feb 6;355(6360):564-6. DOI:10.1038/355564a0 |
5. Macaya RF, Schultze P, Smith FW, Roe JA, and Feigon J. Thrombin-binding DNA aptamer forms a unimolecular quadruplex structure in solution. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3745-9. DOI:10.1073/pnas.90.8.3745 |

Presentations

Most recent (Week 3)

• Week 3 Presentation: Design Progress

Week 2: Original proposal

• Presentation Proposal

Working Team Members

• Tiffany Chan (talk)
• Katherine Fifer (talk)
• Valerie Lau (talk)
• Matthew Meisel (talk)
• ...and others are welcome!