- 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
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
|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)|
- 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 protocol for 10 μL incubation reaction
- 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." 
- Reconstitute 745 NIH units in 98 μL 1% BSA and 882 μL water (0.98 mL total) to give a working stock of 760 units / mL = 650 μg / mL = 10 nmol / mL = 10 μM (formula weight is approximately 65 kDa) 
- In a 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)
- 1 μL of 10 μM thrombin (final concentration: 1 μM = 10 pmol)
- 1 μL of dH2O
- 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 17:52, 10 July 2006 (EDT)
- Schultze P, Macaya RF, and Feigon J. . pmid:8107090.
- Liu Y, Lin C, Li H, and Yan H. . pmid:15945116.
- Li WX, Kaplan AV, Grant GW, Toole JJ, and Leung LL. . pmid:8298130.
- Bock LC, Griffin LC, Latham JA, Vermaas EH, and Toole JJ. . pmid:1741036.
- Macaya RF, Schultze P, Smith FW, Roe JA, and Feigon J. . pmid:8475124.