Biomedical Engineering Reference
In-Depth Information
are a concentration normalized pool from commercial chemical synthesis, while
the 7,308 nt scaffold DNA is derived from a modified genome of the M-13
bacteriophage, cultured and purified within the lab. Once both pools of DNA have
been appropriately prepared, the front and rear nanotube monomers are individually
assembled in separate pools by mixing the scaffold and staple strands together at a
one to six (1:6) ratio in an aqueous buffer with magnesium. Thermal annealing is
performed by briefly heating the mixed pools to 85
ı
C to fully denature all strands
and then gradually cooling down the pools back to room temperature over the course
of 24 h. The sizes of these pools can be easily scaled up or down according to
experimental need. Once the front and rear monomers have assembled, excess staple
material can be removed from solution via a gravity-flow ion-exchange column.
The resulting purified monomer solutions can then be mixed together to allow auto-
heterodimerization to take place. The fully assembled DNA-nanotube heterodimers
are precipitated from solution through PEG precipitation, then rehydrated and
further concentrated for NMR sample preparation.
16.5.2
Preparation of NMR Protein Samples with DNA
Nanotubes
How can membrane proteins be weakly aligned in DNA-nanotube liquid crystals?
Such weak alignment of membrane proteins is achieved by mixing the proteins with
a specific dilution of the DNA liquid crystalline medium such that the interaction
between protein and medium is weak and the protein remains free to tumble
most of the time (Fig.
16.1
c). As is the case with many other media, alignment
occurs due to steric repulsion between the protein and the dilute liquid crystal
medium; charged media can be adjusted for combination of electrostatic and steric
interactions to achieve the same effect. In the case of the DNA nanotube, the mode
of interaction of the protein involves a multimolecular assembly of DNA molecules
through electrostatic interaction between the negatively charged phosphate groups
of DNA and the positively charged amino acids at the surface of the protein. The
aligned NMR sample in presence of DNA nanotubes is simply prepared by manual
mixing of the protein solution with the DNA-nanotube liquid crystal. In practice,
the DNA nanotubes are first reconstituted in the appropriate buffer. Afterwards,
an equivolume sample containing the appropriate amount of protein is added to
the DNA-nanotube solution. The final NMR sample is then prepared by simply
concentrating the sample down to the appropriate sample volume using a common
Centricon concentrator unit. At high concentrations of DNA nanotubes (around
20 mg/mL), the liquid crystal solution will become viscous, an indication that
the sample is ready for RDCs measurements. To reiterate, a DNA-nanotube liquid
crystal with 10% D
2
O yields stable residual quadrupole coupling (RQC) for the
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