Difference between revisions of "IGEM:Harvard/2006/DNA nanostructures"

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(Protocols: redo of protocol to 2 {{ul}} with Dr. Shih's suggestions)
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====Protocols====
 
====Protocols====
  
Potential protocol for 10 {{ul}} incubation reaction
+
Potential protocol for a 2 {{ul}} incubation reaction (revised with Dr. Shih's suggestions)
 
* Reconsitute lyophilized [http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIAL/T6634 bovine thrombin]
 
* Reconsitute lyophilized [http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIAL/T6634 bovine thrombin]
 
** biuret is 745 NIH units = 637 {{ug}} (1170 NIH units = 1 mg)
 
** biuret is 745 NIH units = 637 {{ug}} (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." [http://www.sigmaaldrich.com/sigma-aldrich/product_information_sheet/t6634pis.pdf]
 
** "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." [http://www.sigmaaldrich.com/sigma-aldrich/product_information_sheet/t6634pis.pdf]
** Reconstitute 745 NIH units in 98 {{ul}} 1% BSA and 882 {{ul}} water (0.98 mL total) to give a working stock of 760 units / mL = 650 {{ug}} / mL = 10 nmol / mL = 10 {{um}} (formula weight is approximately 65 kDa) [http://www.sigmaaldrich.com/sigma-aldrich/product_information_sheet/t6634pis.pdf]
+
** 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 {{ug}} / mL = 2 nmol / mL = 2 {{um}} (formula weight is approximately 65 kDa [http://www.sigmaaldrich.com/sigma-aldrich/product_information_sheet/t6634pis.pdf])
 
* In a 0.2 mL PCR tube, mix:
 
* In a 0.2 mL PCR tube, mix:
** 2 {{ul}} of 5x [[IGEM:Harvard/2006/Stock_solutions#Bock.27s_selection_buffer|Bock's selection buffer]]
+
** 0.5 {{ul}} of 4x (not 5x) [[IGEM:Harvard/2006/Stock_solutions#Bock.27s_selection_buffer|Bock's selection buffer]]
** 2 {{ul}} of 10 {{um}} scaffold DNA (final concentration: 2 {{um}} = 20 pmol)
+
** 1.0 {{ul}} of 2 {{um}} aptamers (final concentration: 1.0 {{um}} = 2 pmol)
** 2 {{ul}} of 10 {{um}} oligos (final concentration: 2 {{um}} = 20 pmol)
+
** 0.5 {{ul}} of 2 {{um}} thrombin (final concentration: 0.5 {{um}} = 1 pmol)
** 2 {{ul}} of 10 {{um}} aptamers (final concentration: 2 {{um}} = 20 pmol)
+
* OR in a 0.2 mL PCR tube, mix:
** 1 {{ul}} of 10 {{um}} thrombin (final concentration: 1 {{um}} = 10 pmol)
+
** 0.5 {{ul}} of 4x (not 5x) [[IGEM:Harvard/2006/Stock_solutions#Bock.27s_selection_buffer|Bock's selection buffer]]
** 1 {{ul}} of dH<sub>2</sub>O
+
** 0.5 {{ul}} of 2 {{um}} aptamers (final concentration: 1.0 {{um}} = 1 pmol)
 +
** 1.0 {{ul}} of 2 {{um}} thrombin (final concentration: 0.5 {{um}} = 2 pmol)
 
* Alternative mix: Liu uses 10 pmol of DNA (1 {{ul}} of 10 {{um}}) and varies thrombin amount from 2 pmol (1 {{ul}} of 0.2x thrombin working stock) to 100 pmol (1 {{ul}} of 10x thrombin working stock)
 
* Alternative mix: Liu uses 10 pmol of DNA (1 {{ul}} of 10 {{um}}) and varies thrombin amount from 2 pmol (1 {{ul}} of 0.2x thrombin working stock) to 100 pmol (1 {{ul}} of 10x thrombin working stock)
 
* Incubate at room temperature for 30 min.
 
* Incubate at room temperature for 30 min.
 
* Load onto a non-denaturing polyacrylamide gel (10% to 20% gradient)
 
* Load onto a non-denaturing polyacrylamide gel (10% to 20% gradient)
 
** Liu runs at 25 mA for 48 h.
 
** Liu runs at 25 mA for 48 h.
[[User:Matthewmeisel|Matthewmeisel]] 17:52, 10 July 2006 (EDT)
+
[[User:Matthewmeisel|Matthewmeisel]] 11:11, 11 July 2006 (EDT)
  
 
====Bibliography====
 
====Bibliography====

Revision as of 07:11, 11 July 2006

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

IGEM harv06 mattspecs.gif

Container Designs

Latch Designs

Coding

Existing code

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 | PubMed ID:8107090 | HubMed [tha1]
  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 | PubMed ID:15945116 | HubMed [tha2]
  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. PubMed ID:8298130 | HubMed [tha3]
  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 | PubMed ID:1741036 | HubMed [tha4]
  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. PubMed ID:8475124 | HubMed [tha5]
All Medline abstracts: PubMed | HubMed

Presentations

Most recent (Week 3)

Week 2: Original proposal

Working Team Members